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Granulomatosis with polyangiitis (GPA) is a rare but serious necrotizing auto-immune vasculitis. GPA is mostly associated with the presence of Anti-Neutrophil Cytoplasmic Antibody (ANCA) targeting proteinase 3 (PR3-ANCA), a serine protease contained in neutrophil granules but also exposed at the membrane. PR3-ANCAs have a proven fundamental role in GPA: they bind neutrophils allowing their auto-immune activation responsible for vasculitis lesions. PR3-ANCAs bind neutrophil surface on the one hand by their Fab binding PR3 and on the other by their Fc binding Fc gamma receptors. Despite current therapies, GPA is still a serious disease with an important mortality and a high risk of relapse. Furthermore, although PR3-ANCAs are a consistent biomarker for GPA diagnosis, relapse management currently based on their level is inconsistent. Indeed, PR3-ANCA level is not correlated with disease activity in 25% of patients suggesting that not all PR3-ANCAs are pathogenic. Therefore, the development of new biomarkers to evaluate disease activity and predict relapse and new therapies is necessary. Understanding factors influencing PR3-ANCA pathogenicity, i.e. their potential to induce auto-immune activation of neutrophils, offers interesting perspectives in order to improve GPA management. Most relevant factors influencing PR3-ANCA pathogenicity are involved in their interaction with neutrophils: level of PR3 autoantigen at neutrophil surface, epitope of PR3 recognized by PR3-ANCA, isotype and glycosylation of PR3-ANCA. We detailed in this review the advances in understanding these factors influencing PR3-ANCA pathogenicity in order to use them as biomarkers and develop new therapies in GPA as part of a personalized approach. [ABSTRACT FROM AUTHOR]

Polymorphonuclear neutrophils contain at least four serine endopeptidases, namely neutrophil elastase (NE), proteinase 3 (PR3), cathepsin G (CatG), and NSP4, which contribute to the regulation of infection and of inflammatory processes. In physiological conditions, endogenous inhibitors including α2‐macroglobulin (α2‐M), serpins [α1‐proteinase inhibitor (α1‐PI)], monocyte neutrophil elastase inhibitor (MNEI), α1‐antichymotrypsin, and locally produced chelonianins (elafin, SLPI) control excessive proteolytic activity of neutrophilic serine proteinases. In contrast to human NE (hNE), hPR3 is weakly inhibited by α1‐PI and MNEI but not by SLPI. α2‐M is a large spectrum inhibitor that traps a variety of proteinases in response to cleavage(s) in its bait region. We report here that α2‐M was more rapidly processed by hNE than hPR3 or hCatG. This was confirmed by the observation that the association between α2‐M and hPR3 is governed by a kass in the ≤ 105 m−1·s−1 range. Since α2‐M‐trapped proteinases retain peptidase activity, we first predicted the putative cleavage sites within the α2‐M bait region (residues 690–728) using kinetic and molecular modeling approaches. We then identified by mass spectrum analysis the cleavage sites of hPR3 in a synthetic peptide spanning the 39‐residue bait region of α2‐M (39pep‐α2‐M). Since the 39pep‐α2‐M peptide and the corresponding bait area in the whole protein do not contain sequences with a high probability of specific cleavage by hPR3 and were indeed only slowly cleaved by hPR3, it can be concluded that α2‐M is a poor inhibitor of hPR3. The resistance of hPR3 to inhibition by endogenous inhibitors explains at least in part its role in tissue injury during chronic inflammatory diseases and its well‐recognized function of major target autoantigen in granulomatosis with polyangiitis. [ABSTRACT FROM AUTHOR]

Background: The pathophysiology of subclinical versus clinical rejection remains incompletely understood given their equivalent histological severity but discordant graft function. The goal was to evaluate serine hydrolase enzyme activities to explore if there were any underlying differences in activities during subclinical versus clinical rejection. Methods: Serine hydrolase activity-based protein profiling (ABPP) was performed on the urines of a case control cohort of patients with biopsy confirmed subclinical or clinical transplant rejection. In-gel analysis and affinity purification with mass spectrometry were used to demonstrate and identify active serine hydrolase activity. An assay for proteinase 3 (PR3/PRTN3) was adapted for the quantitation of activity in urine. Results: In-gel ABPP profiles suggested increased intensity and diversity of serine hydrolase activities in urine from patients undergoing subclinical versus clinical rejection. Serine hydrolases (n = 30) were identified by mass spectrometry in subclinical and clinical rejection patients with 4 non-overlapping candidates between the two groups (i.e. ABHD14B, LTF, PR3/PRTN3 and PRSS12). Western blot and the use of a specific inhibitor confirmed the presence of active PR3/PRTN3 in samples from patients undergoing subclinical rejection. Analysis of samples from normal donors or from several serial post-transplant urines indicated that although PR3/PRTN3 activity may be highly associated with low-grade subclinical inflammation, the enzyme activity was not restricted to this patient group. Conclusions: There appear to be limited qualitative and quantitative differences in serine hydrolase activity in patients with subclinical versus clinical renal transplant rejection. The majority of enzymes identified were present in samples from both groups implying that in-gel quantitative differences may largely relate to the activity status of shared enzymes. However qualitative compositional differences were also observed indicating differential activities. The PR3/PRTN3 analyses indicate that the activity status of urine in transplant patients is dynamic possibly reflecting changes in the underlying processes in the transplant. These data suggest that differential serine hydrolase pathways may be active in subclinical versus clinical rejection which requires further exploration in larger patient cohorts. Although this study focused on PR3/PRTN3, this does not preclude the possibility that other enzymes may play critical roles in the rejection process. [ABSTRACT FROM AUTHOR]

Cathepsin C (CatC, syn. Dipeptidyl peptidase I) is a lysosomal cysteine proteinase expressed in several tissues including inflammatory cells. This enzyme is important for maintaining multiple cellular functions and for processing immune cell-derived proteases. While mutations in the CatC gene were reported in Papillon-Lefèvre syndrome, a rare autosomal recessive disorder featuring hyperkeratosis and periodontitis, evidence from clinical and preclinical studies points toward pro-inflammatory effects of CatC in various disease processes that are mainly mediated by the activation of neutrophil serine proteinases. Moreover, tumor-promoting effects were ascribed to CatC. The aim of this review is to highlight current knowledge of the CatC as a potential therapeutic target in inflammatory disorders. [Display omitted] • Cathepsin C activates granule serine proteinases involved in inflammation. • Cathepsin C contributes to the pathogenesis of different acute and chronic diseases. • Potential of cathepsin C inhibition to ameliorate inflammation in preclinical models. • Need for studies evaluating CatC inhibitors repurposing in chronic inflammatory diseases. [ABSTRACT FROM AUTHOR]

Neutrophils are one of the most important military services of the armed forces of the immune system, a crucial line of defense against bacterial or fungal onslaughts. One of their mechanisms of action relies on the production of serine proteases. One of these enzymes is proteinase 3 (PR3), which is engaged in the processing of pro-inflammatory cytokines, receptors, heat shock proteins and in the generation of antibacterial peptides. Despite its protective function, uncontrolled activity of PR3 has been associated with the progression of inflammation and tissue injury. Although PR3 was characterized at the beginning of 90's of the last century for the first time, the research on the development of its inhibitors is barely noticeable. Here we present the recent findings on the design, synthesis and the activity of phosphonic PR3 inhibitors together with the historical perspective. Image 1 • Proteinase 3 is considered an interesting drug target since its activity has been associated with different pathophysiological disorders. • Several classes of PR3 inhibitors such as isocoumarins, N -hydroxysuccinimides, thiatriazolidine and phosphonates have been developed. • Diaryl esters of phosphonic analogues of amino acids represent an interesting class of irreversible inhibitors of proteinase 3. • Phosphonic inhibitors can be easily turned into the activity-based probes useful for protease visualization either in vitro or in cell assays. [ABSTRACT FROM AUTHOR]

Proteinase 3 (PR3) is the main target antigen of antineutrophil cytoplasmic antibodies (ANCAs) in PR3-ANCA-associated vasculitis. A small fraction of PR3 is constitutively exposed on the surface of quiescent blood neutrophils in a proteolytically inactive form. When activated, neutrophils expose an induced form of membrane-bound PR3 (PR3mb) on their surface as well, which is enzymatically less active than unbound PR3 in solution due to its altered conformation. In this work, our objective was to understand the respective role of constitutive and induced PR3mb in the immune activation of neutrophils triggered by murine anti-PR3 mAbs and human PR3-ANCA. We quantified immune activation of neutrophils by the measurement of the production of superoxide anions and secreted protease activity in the cell supernatant before and after treatment of the cells by alpha-1 protease inhibitor that clears induced PR3mb from the cell surface. Incubation of TNFa-primed neutrophils with anti-PR3 antibodies resulted in a significant increase in superoxide anion production, membrane activation marker exposition, and secreted protease activity. When primed neutrophils were first treated with alpha-1 protease inhibitor, we observed a partial reduction in antibody-induced neutrophil activation, suggesting that constitutive PR3mb is sufficient to activate neutrophils. The pretreatment of primed neutrophils with purified antigenbinding fragments used as competitor significantly reduced cell activation by whole antibodies. This led us to the conclusion that PR3mb promoted immune activation of neutrophils. We propose that blocking and/or elimination of PR3mb offers a new therapeutic strategy to attenuate neutrophil activation in patients with PR3-ANCA-associated vasculitis. [ABSTRACT FROM AUTHOR]

Background Vascular damage and primary graft dysfunction increase with prolonged preservation times of transplanted donor lungs. Hence, storage and conservation of donated lungs in protein-free, dextran-containing electrolyte solutions, like Perfadex, is limited to about 6 hours. We hypothesized that transplanted lungs are protected against neutrophil-mediated proteolytic damage by adding α 1 -anti-trypsin (AAT), a highly abundant human plasma proteinase inhibitor, to Perfadex. Methods A realistic clinically oriented murine model of lung transplantation was used to simulate the ischemia–reperfusion process. Lung grafts were stored at 4°C in Perfadex solution supplemented with AAT or an AAT mutant devoid of elastase-inhibiting activity for 18 hours. We examined wild-type and proteinase 3/neutrophil elastase (PR3/NE) double-deficient mice as graft recipients. Gas exchange function and infiltrating neutrophils of the transplanted lung, as well as protein content and neutrophil numbers in the bronchoalveolar lavage fluid, were determined. Results AAT as a supplement to Perfadex reduced the extent of primary graft dysfunction and early neutrophil responses after extended storage for 18 hours at 4°C and 4-hour reperfusion in the recipients. Double-knockout recipients that lack elastase-like activities in neutrophils were also protected from early reperfusion injury, but not lung grafts that were perfused with a reactive center mutant of AAT devoid of elastase-inhibiting activity. Conclusions PR3 and NE, the principal targets of AAT, are major triggers of post-ischemic reperfusion damage. Their effective inhibition in the graft and recipient is a promising strategy for organ usage after storage for >6 hours. [ABSTRACT FROM AUTHOR]