Targeting Cathepsin C in PR3-ANCA Vasculitis.

BACKGROUND: The ANCA autoantigens proteinase 3 (PR3) and myeloperoxidase (MPO) are exclusively expressed by neutrophils and monocytes. ANCA-mediated activation of these cells is the key driver of the vascular injury process in ANCA-associated vasculitis (AAV), and neutrophil serine proteases (NSPs) are disease mediators. Cathepsin C (CatC) from zymogens activates the proteolytic function of NSPs, including PR3. Lack of NSP zymogen activation results in neutrophils with strongly reduced NSP proteins. METHODS: To explore AAV-relevant consequences of blocking NSP zymogen activation by CatC, we used myeloid cells from patients with Papillon-Lefèvre syndrome, a genetic deficiency of CatC, to assess NSPs and NSP-mediated endothelial cell injury. We also examined pharmacologic CatC inhibition in neutrophil-differentiated human hematopoietic stem cells, primary human umbilical vein cells, and primary glomerular microvascular endothelial cells. RESULTS: Patients with Papillon-Lefèvre syndrome showed strongly reduced NSPs in neutrophils and monocytes. Neutrophils from these patients produced a negative PR3-ANCA test, presented less PR3 on the surface of viable and apoptotic cells, and caused significantly less damage in human umbilical vein cells. These findings were recapitulated in human stem cells, in which a highly specific CatC inhibitor, but not prednisolone, reduced NSPs without affecting neutrophil differentiation, reduced membrane PR3, and diminished neutrophil activation upon PR3-ANCA but not MPO-ANCA stimulation. Compared with healthy controls, neutrophils from patients with Papillon-Lefèvre syndrome transferred less proteolytically active NSPs to glomerular microvascular endothelial cells, the cell type targeted in ANCA-induced necrotizing crescentic glomerulonephritis. Finally, both genetic CatC deficiency and pharmacologic inhibition, but not prednisolone, reduced neutrophil-induced glomerular microvascular endothelial cell damage. CONCLUSIONS: These findings may offer encouragement for clinical studies of adjunctive CatC inhibitor in patients with PR3-AAV.

Consequences of cathepsin C inactivation for 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, a systemic small-vessel vasculitis. Binding of ANCA to PR3m 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 pro-forms 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 therefore has implications as a novel therapeutic approach in granulomatosis with polyangiitis. 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 with 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 were 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 stability 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.

Endothelial NF-κB Blockade Abrogates ANCA-Induced GN.

ANCA-associated vasculitis (AAV) is a highly inflammatory condition in which ANCA-activated neutrophils interact with the endothelium, resulting in necrotizing vasculitis. We tested the hypothesis that endothelial NF-κB mediates necrotizing crescentic GN (NCGN) and provides a specific treatment target. Reanalysis of kidneys from previously examined murine NCGN disease models revealed NF-κB activation in affected kidneys, mostly as a p50/p65 heterodimer, and increased renal expression of NF-κB-dependent tumor necrosis factor α (TNF-α). NF-κB activation positively correlated with crescent formation, and nuclear phospho-p65 staining showed NF-κB activation within CD31-expressing endothelial cells (ECs) in affected glomeruli. Therefore, we studied the effect of ANCA on NF-κB activation in neutrophil/EC cocultures in vitro ANCA did not activate NF-κB in primed human neutrophils, but ANCA-stimulated primed neutrophils activated NF-κB in ECs, at least in part via TNF-α release. This effect increased endothelial gene transcription and protein production of NF-κB-regulated interleukin-8. Moreover, upregulation of endothelial NF-κB promoted neutrophil adhesion to EC monolayers, an effect that was inhibited by a specific IKKß inhibitor. In a murine NCGN model, prophylactic application of E-selectin-targeted immunoliposomes packed with p65 siRNA to downregulate endothelial NF-κB significantly reduced urine abnormalities, renal myeloid cell influx, and NCGN. Increased glomerular endothelial phospho-p65 staining in patients with AAV indicated that NF-κB is activated in human NCGN also. We suggest that ANCA-stimulated neutrophils activate endothelial NF-κB, which contributes to NCGN and provides a potential therapeutic target in AAV.

Protective α1-antitrypsin effects in autoimmune vasculitis are compromised by methionine oxidation.

BackgroundAntineutrophil cytoplasmic autoantibody-associated (ANCA-associated) vasculitidies (AAV) are life-threatening systemic autoimmune conditions. ANCAs directed against proteinase 3 (PR3) or myeloperoxidase (MPO) bind their cell surface-presented antigen, activate neutrophils, and cause vasculitis. An imbalance between PR3 and its major inhibitor α1-antitrypsin (AAT) was proposed to underlie PR3- but not MPO-AAV. We measured AAT and PR3 in healthy individuals and patients with AAV and studied protective AAT effects pertaining to PR3- and MPO-ANCA.MethodsPlasma and blood neutrophils were assessed for PR3 and AAT. WT, mutant, and oxidation-resistant AAT species were produced to characterize AAT-PR3 interactions by flow cytometry, immunoblotting, fluorescence resonance energy transfer assays, and surface plasmon resonance measurements. Neutrophil activation was measured using the ferricytochrome C assay and AAT methionine-oxidation by Parallel Reaction Monitoring.ResultsWe found significantly increased PR3 and AAT pools in patients with both PR3- and MPO-AAV; however, only in PR3-AAV did the PR3 pool correlate with the ANCA titer, inflammatory response, and disease severity. Mechanistically, AAT prevented PR3 from binding to CD177, thereby reducing neutrophil surface antigen for ligation by PR3-ANCA. Active patients with PR3-AAV showed critical methionine-oxidation in plasma AAT that was recapitulated by ANCA-activated neutrophils. The protective PR3-related AAT effects were compromised by methionine-oxidation in the AAT reactive center loop but preserved when 2 critical methionines were substituted with valine and leucine.ConclusionPathogenic differences between PR3- and MPO-AAV are related to AAT regulation of membrane-PR3, attenuating neutrophil activation by PR3-ANCA rather than MPO-ANCA. Oxidation-resistant AAT could serve as adjunctive therapy in PR3-AAV.FUNDINGThis work was supported by KE 576/10-1 from the Deutsche Forschungsgemeinschaft, SCHR 771/8-1 from the Deutsche Forschungsgemeinschaft, grant 394046635 - SFB 1365 from the Deutsche Forschungsgemeinschaft, and ECRC grants.

Gene silencing and a novel monoallelic expression pattern in distinct CD177 neutrophil subsets.

CD177 presents antigens in allo- and autoimmune diseases on the neutrophil surface. Individuals can be either CD177-deficient or harbor distinct CD177neg and CD177pos neutrophil subsets. We studied mechanisms controlling subset-restricted CD177 expression in bimodal individuals. CD177pos, but not CD177neg neutrophils, produced CD177 protein and mRNA. Haplotype analysis indicated a unique monoallelic CD177 expression pattern, where the offspring stably transcribed either the maternal or paternal allele. Hematopoietic stem cells expressed both CD177 alleles and silenced one copy during neutrophil differentiation. ChIP and reporter assays in HeLa cells with monoallelic CD177 expression showed that methylation reduced reporter activity, whereas demethylation caused biallelic CD177 expression. HeLa cell transfection with c-Jun and c-Fos increased CD177 mRNA. Importantly, CD177pos human neutrophils, but not CD177neg neutrophils, showed a euchromatic CD177 promoter, unmethylated CpGs, and c-Jun and c-Fos binding. We describe epigenetic mechanisms explaining the two distinct CD177 neutrophil subsets and a novel monoallelic CD177 expression pattern that does not follow classical random monoallelic expression or imprinting.

Lessons from a double-transgenic neutrophil approach to induce antiproteinase 3 antibody-mediated vasculitis in mice.

ANCA to either PR3 or MPO are found in patients with necrotizing vasculitis and glomerulonephritis. ANCA binding to their target antigens on neutrophils and subsequent neutrophil activation are pivotal disease mechanisms that lead to vascular inflammation and necrosis. ANCA interaction with PR3 is more complex than with MPO as the neutrophil-specific CD177 receptor is involved in PR3 surface expression and PR3-ANCA-induced neutrophil activation. Modeling human disease is important to clinical research. Highly successful mouse models of MPO-ANCA vasculitis exist; however, recapitulating PR3-ANCA vasculitis has not been successful. We generated double-transgenic (DT) mice that expressed human PR3 and CD177 under a myeloid-specific huMRP8 promoter in an attempt to model PR3-ANCA vasculitis. DT mice strongly expressed the human transgenes in and on murine neutrophils and bound murine and human anti-PR3 antibodies. Nevertheless, passive transfer of these antibodies into LPS-primed DT mice or immunization of C57BL/6 mice with human PR3 followed by irradiation and transplantation of DT bone marrow failed to induce glomerulonephritis. Further analyses revealed that anti-PR3 antibodies did not activate DT neutrophils as shown by superoxide generation. Moreover, we found that mice did not properly process human pro-PR3 into mature PR3 and, consequently, the signaling complex between PR3, CD177, and CD11b, which promotes neutrophil activation by anti-PR3 antibodies, failed to form. We conclude that important species differences in PR3 and CD177 exist between men and mice that prevented successful generation of a murine anti-PR3 antibody model.