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In alpha-1-antitrypsin deficiency (AATD), neutrophil serine proteases such as elastase and proteinase 3 (PR3) are insufficiently inhibited. A previous study in AATD patients showed a higher plasma level of the specific PR3-generated fibrinogen-derived peptide AαVal541, compared with healthy controls. Here, we analyzed the course of AαVal541 plasma levels during 4 weeks after a single iv dose of 240 mg/kg AAT in ten patients with genotype Z/Rare or Rare/Rare. To this end, we developed an immunoassay to measure AαVal541 in plasma and applied population pharmacokinetic modeling for AAT. The median AαVal541 plasma level before treatment was 140.2 nM (IQR 51.5-234.8 nM)). In five patients who received AAT for the first time, AαVal541 levels decreased to 20.6 nM (IQR 5.8-88.9 nM), and in five patients who already had received multiple infusions before, it decreased to 26.2 nM (IQR 22.31-35.0 nM). In 9 of 10 patients, AαVal541 levels were reduced to the median level of healthy controls (21.4 nM; IQR 16.7-30.1 nM). At 7-14 days after treatment, AαVal541 levels started to increase again in all patients. Our results show that fibrinopeptide AαVal541 may serve as a biochemical footprint to assess the efficacy of in vivo inhibition of PR3 activity in patients receiving intravenous AAT augmentation therapy.

Background and Objectives: Rituximab is approved in patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) and leads to a decrease of ANCA levels. The objectives of this study were to investigate the non-linear pharmacokinetics of rituximab and the relationship between its concentrations and ANCA levels in AAV patients., Methods: Ninety-two AAV patients from the RAVE (Rituximab in ANCA-Associated Vasculitis) trial were assessed. Both ANCA anti-myeloperoxidase (MPO-ANCA) and anti-proteinase 3 (PR3-ANCA) levels were used as biomarkers. The pharmacokinetics of rituximab were described using a semi-mechanistic two-compartment model that included a latent target antigen turnover and allowed the estimation of specific target-mediated elimination in addition to its non-specific elimination of rituximab. The effect of rituximab on the ANCA level was described using a semi-mechanistic compartment model with a negative feedback (Friberg) model with no transit compartment. A population modeling approach was used., Results: Our pharmacokinetic and pharmacokinetic-pharmacodynamic (PK-PD) models satisfactorily described both concentration-time and concentration-effect relationship data. The mean (inter-individual standard deviation) estimated non-specific clearance was 0.15 L/day (0.30%) and the target-mediated elimination rate constant was 2.4 × 10 -5 nmol/day. The elimination half-lives for MPO-ANCA and PR3-ANCA were 24 and 18 days, respectively., Conclusions: A non-linear target-mediated elimination of rituximab was detected in AAV patients. Our PK-PD model allowed quantification of the association between rituximab concentrations and ANCA levels. This decrease was deep but delayed, and more sustained in patients with MPO-ANCA than in those with PR3-ANCA. Our results suggest that repeating courses of rituximab might improve the clinical response to rituximab.

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., (Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

IκBα protein plays an important role in NFκB signaling pathway regulation. The dysfunction of IκBα is tightly related to various diseases, including cancers. However, the molecular mechanisms by which IκBα loses its normal functions are diverse and complex. Here, we reported a novel cleavage of IκBα protein occurred in AML cells. Compared with the full-length IκBα protein, the truncated IκBα fragment exhibited a dramatically weak binding ability to NFκB complex and showed a significant decreased inhibition on NFκB transactivation. Knockdown of PR3, a serine protease mainly expressed in myeloid cells, could inhibit such IκBα cleavage and enhance the sensitivities of AML cells to the differentiation inducers. In addition, we showed that the level of PR3 mRNA was relatively higher in newly diagnosed AML patients than in those patients with complete remission, suggesting that PR3 expression and its involvement in IκBα cleavage might be closely associated with AML. Our studies revealed for the first time a PR3-involved IκBα cleavage in AML cells, providing some new evidences for further understanding the mechanisms underlying the deregulation of NFκB pathway in AML. Finally, we also suggested a potential clinical application value of PR3 protein in the treatment and prognosis surveillance for leukemia., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Anti-neutrophil cytoplasmic autoantibodies (ANCA) directed against myeloperoxidase (MPO) and proteinase 3 (PR3) are pathogenic in ANCA-associated vasculitis (AAV). The respective role of IgG Fc and Fab glycosylation in mediating ANCA pathogenicity is incompletely understood. Herein we investigate in detail the changes in Fc and Fab glycosylation in MPO-ANCA and Pr3-ANCA and examine the association of glycosylation aberrancies with disease activity., Methodology: Total IgG was isolated from serum or plasma of a cohort of 30 patients with AAV (14 MPO-ANCA; 16 PR3-ANCA), and 19 healthy control subjects. Anti-MPO specific IgG was affinity-purified from plasma of an additional cohort of 18 MPO-ANCA patients undergoing plasmapheresis. We used lectin binding assays, liquid chromatography, and mass spectrometry-based methods to analyze Fc and Fab glycosylation, the degree of sialylation of Fc and Fab fragments and to determine the exact localization of N-glycans on Fc and Fab fragments., Principal Findings: IgG1 Fc glycosylation of total IgG was significantly reduced in patients with active AAV compared to controls. Clinical remission was associated with complete glycan normalization for PR3-ANCA patients but not for MPO-ANCA patients. Fc-glycosylation of anti-MPO specific IgG was similar to total IgG purified from plasma. A major fraction of anti-MPO specific IgG harbor extensive glycosylation within the variable domain on the Fab portion., Conclusions/significance: Significant differences exist between MPO and PR3-ANCA regarding the changes in amounts and types of glycans on Fc fragment and the association with disease activity. These differences may contribute to significant clinical difference in the disease course observed between the two diseases., Competing Interests: The authors have declared that no competing interests exist.

Background: A recent study found that CD177 served as a receptor of membrane-bound proteinase-3 (mPR3) in a subset of neutrophils. Furthermore, CD177 has been identified as a high-affinity heterophilic binding partner for the endothelial cell platelet endothelial cell adhesion molecule-1 (PECAM-1). The current study aimed to investigate whether the interaction between PECAM-1 and CD177 could influence mPR3 expression as well as PR3-antineutrophil cytoplasmic antibody (ANCA)-induced neutrophil activation and glomerular endothelial cell (GEnC) injury., Methods: The effect of interaction between CD177 and PECAM-1 on mPR3 expression was explored by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The effect of PECAM-1 on neutrophil activation and GEnC injury induced by PR3-ANCA-positive immunoglobulin (Ig)Gs was evaluated by dihydrorhodamine (DHR) assay and ELISA. CD177-negative neutrophils were selected by magnetic cell sorting (MACS), and the inhibitory effect of PECAM-1 on CD177-negative and mixed neutrophils was explored by measuring neutrophil degranulation., Results: The level of specific interaction between CD177 and PECAM-1 was elevated with increasing CD177 concentration. The expression of mPR3 significantly decreased in neutrophils preincubated with PECAM-1 in a dose-dependent manner. Consistently, the levels of respiratory burst and degranulation induced by PR3-ANCA-positive IgGs in recombinant human tumor necrosis factor-alpha (TNF-α)-primed neutrophils was significantly reduced by preincubation with PECAM-1 (440.6 ± 123.0 vs. 511.4 ± 95.5, p < 0.05; and 3155.0 ± 1733.0 ng/ml vs. 5903.0 ± 717.5 ng/ml, p < 0.05, respectively). In CD177-negative neutrophils incubated with PR3-ANCA-positive IgGs, the level of degranulation was not significantly changed by preincubation with PECAM-1. However, in mixed neutrophils, PECAM-1 significantly decreased the level of degranulation induced by PR3-ANCA-positive IgGs (1015.9 ± 229.2% vs. 1725.2 ± 412.4%, p < 0.01). Furthermore, with preincubation of TNF-α-primed neutrophils with PECAM-1, the level of soluble intercellular cell adhesion molecule-1 (sICAM-1), a marker of endothelial cell activation and injury, in the supernatant of GEnCs treated with primed neutrophils plus PR3-ANCA-positive IgGs was significantly attenuated (112.7 ± 24.2 pg/ml vs. 167.5 ± 27.7 pg/ml, p < 0.05)., Conclusions: PECAM-1 can decrease the level of mPR3 expression on neutrophils, resulting in attenuation of neutrophil activation and subsequent GEnC injury induced by PR3-ANCA-positive IgGs.

Chronic Obstructive Pulmonary Disease (COPD) is a common, multifactorial lung disease which results in significant impairment of patients' health and a large impact on society and health care burden. It is believed to be the result of prolonged, destructive neutrophilic inflammation which results in progressive damage to lung structures. During this process, large quantities of neutrophil serine proteinases (NSPs) are released which initiate the damage and contribute towards driving a persistent inflammatory state.Neutrophil elastase has long been considered the key NSP involved in the pathophysiology of COPD. However, in recent years, a significant role for Proteinase 3 (PR3) in disease development has emerged, both in COPD and other chronic inflammatory conditions. Therefore, there is a need to investigate the importance of PR3 in disease development and hence its potential as a therapeutic target. Research into PR3 has largely been confined to its role as an autoantigen, but PR3 is involved in triggering inflammatory pathways, disrupting cellular signalling, degrading key structural proteins, and pathogen response.This review summarises what is presently known about PR3, explores its involvement particularly in the development of COPD, and indicates areas requiring further investigation.

BACKGROUND Systemic vasculitis can present with a multitude of symptoms involving multiple organ systems. Clinicians should avoid anchoring bias and be cognizant that different types of vasculitides can be present in the same patient and that the diagnosis of one should not preclude the subsequent diagnosis of another. CASE REPORT A 67-year-old woman was referred for evaluation of episodes of epistaxis and recurrent severe sinusitis. Her physical examination showed nasal congestion and purpuric rash on the lower extremities. CT of the sinuses showed severe mucosal thickening. ANCA serologies were positive with a c-ANCA titer of 1: 5120 and anti-proteinase-3 (anti-PR3) antibodies of 1061 units. Serum creatinine was elevated at 1.32 mg/dL (GFR of 40.62 ml/min). Urine analysis showed proteinuria and hematuria. The patient declined treatment initially, but while awaiting kidney biopsy she developed episodes of headache and blurry vision. She underwent right temporal artery biopsy 4 days later, which confirmed the diagnosis of GCA. The biopsy showed characteristic histopathology findings and she was started on 60 mg of prednisone daily. The kidney biopsy showed pauci-immune crescentic glomerulonephritis (PICGN) consistent with ANCA-associated vasculitis. We identified all the cases of co-presentation of GCA and GPA in the literature and summarized their clinical features in this report. CONCLUSIONS Astute clinicians should be cognizant of overlapping and atypical presentations of vasculitides to avoid delayed diagnosis and errors in management.

The small vessel vasculitides granulomatosis with polyangiitis (GPA) and microscopic polyangiitis are associated with autoantibodies to neutrophil cytoplasm antigens (ANCA), principally proteinase-3 (PR3) and myeloperoxidase (MPO). There is an association between GPA and nasal carriage of Staphylococcus aureus. The recent finding that S. aureus produces proteins that bind tightly to and block the function of both PR3 and MPO suggests a mechanism for ANCA formation. The bacterial protein-autoantigen conjugate is recognised by B cells with ANCA specificity, internalised, and the bacterial protein processed and presented to T cells with specificity for bacterial peptides. The T cell can then provide help to the B cell, allowing class switching, affinity maturation and the production of pathogenic ANCA. This mechanism predicts that T cells with this specificity will be found in patients, and that the bacterial protein-autoantigen conjugate will be particularly efficient at eliciting ANCA production., (Crown Copyright © 2018. Published by Elsevier Ltd. All rights reserved.)

Background: One of characteristic findings in granulomatosis with polyangiitis (GPA) is the presence of proteinase-3 (anti-PR3) specific antibodies. These antibodies can cause neutrophil activation, degranulation and generation of reactive oxygen species (ROS). Each of these inflammatory events can be suppressed by circulating alpha-1 antitrypsin (A1AT). A1AT is an acute phase protein increasing during inflammation, however, it may circulate as an inactive polymeric protein. The aim was to analyze how different types of A1AT can affect anti-PR3 mediated neutrophil activation., Methods: Granulocytes were obtained from the blood of healthy volunteers and purified by density gradient centrifugation. Effects of A1AT on IgG anti-PR3-mediated neutrophil activation were evaluated by stimulation of the cells with native IgG anti-PR3 antibodies in the presence of native or polymerized A1AT. Analyses of selected proinflammatory genes expression were performed using quantitative real-time. Flow cytometry was used to study the cell membrane PR3, its binding by anti-PR3 IgG, and production of ROS at presence A1AT. Neutrophil elastase complexes with A1AT were measured by ELISA., Results: Native A1AT inhibited formation of the immune complex of PR3 with anti-PR3 and anti-PR3-mediated neutrophil activation/ROS production. Protective effect of polymerized A1AT against these events was diminished at least fivefold., Conclusions: Native A1AT can prevent pivotal events of neutrophils' activation by anti-PR3 IgG, the main autoantibody in anti-PR3 dependent vasculitis. Inhibitory properties of polymerized A1AT, decreased plausibly due to a loss of anti-protease function, can explain more severe course of the disease in subjects with deficiency of A1AT., (Copyright © 2016. Published by Elsevier Urban & Partner Sp. z o.o.)

The balance between neutrophil serine proteases (NSPs) and protease inhibitors (PIs) in the lung is a critical determinant for a number of chronic inflammatory lung diseases such as chronic obstructive pulmonary disease, cystic fibrosis and acute lung injury. During activation at inflammatory sites, excessive release of NSPs such as human neutrophil elastase (HNE), proteinase 3 (Pr3) and cathepsin G (CatG), leads to destruction of the lung matrix and continued propagation of acute inflammation. Under normal conditions, PIs counteract these effects by inactivating NSPs; however, in chronic inflammatory lung diseases, there are insufficient amounts of PIs to mitigate damage. Therapeutic strategies are needed to modulate excessive NSP activity for the clinical management of chronic inflammatory lung diseases. In the study reported here, a panel of N-arylacyl O-sulfonated aminoglycosides was screened to identify inhibitors of the NSPs. Dose-dependent inhibitors for each individual serine protease were identified. Select compounds were found to inhibit multiple NSPs, including one lead structure that is shown to inhibit all three NSPs. Two lead compounds identified during the screen for each individual NSP were further characterized as partial mixed inhibitors of CatG. Concentration-dependent inhibition of protease-mediated detachment of lung epithelial cells is demonstrated., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Proteinase 3 (PR3) has received great scientific attention after its identification as the essential antigenic target of antineutrophil cytoplasm antibodies in Wegener's granulomatosis (now called granulomatosis with polyangiitis). Despite many structural and functional similarities between neutrophil elastase (NE) and PR3 during biosynthesis, storage, and extracellular release, unique properties and pathobiological functions have emerged from detailed studies in recent years. The development of highly sensitive substrates and inhibitors of human PR3 and the creation of PR3-selective single knockout mice led to the identification of nonredundant roles of PR3 in cell death induction via procaspase-3 activation in cell cultures and in mouse models. According to a study in knockout mice, PR3 shortens the lifespan of infiltrating neutrophils in tissues and accelerates the clearance of aged neutrophils in mice. Membrane exposure of active human PR3 on apoptotic neutrophils reprograms the response of macrophages to phagocytosed neutrophils, triggers secretion of proinflammatory cytokines, and undermines immune silencing and tissue regeneration. PR3-induced disruption of the anti-inflammatory effect of efferocytosis may be relevant for not only granulomatosis with polyangiitis but also for other autoimmune diseases with high neutrophil turnover. Inhibition of membrane-bound PR3 by endogenous inhibitors such as the α-1-protease inhibitor is comparatively weaker than that of NE, suggesting that the adverse effects of unopposed PR3 activity resurface earlier than those of NE in individuals with α-1-protease inhibitor deficiency. Effective coverage of PR3 by anti-inflammatory tools and simultaneous inhibition of both PR3 and NE should be most promising in the future., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Proteinase 3 (PR3) is released from neutrophil granules and is involved in the inflammatory process. PR3 is implicated in antimicrobial defense and cell death, but the exact role of PR3 in the brain is less defined. Microglia is the major immune effector cells in the CNS and is activated by brain injury. In the present study, the effect of PR3 on glial activation was investigated. Microglial activation was assessed by the intracellular level of reactive oxygen species and expression of inflammatory cytokines. The conditioned media from activated microglia by PR3 was used for measuring the neurotoxic effects of PR3-stimulated microglia. The effects of PR3 in vivo were measured by microinjecting PR3 into the rat brain. Herein we show that PR3 increased the inflammatory responses including the intracellular ROS and pro-inflammatory cytokine production in rat primary microglia. Conditioned media from PR3-treated microglia induced neuronal cell death in a concentration dependent manner. Furthermore, microinjected PR3 into the striatum of the rat brain induced microglial activation and neuronal cell death. Interestingly treatment with anti-PR3 monoclonal antibody and protease inhibitors ameliorated microglial activation induced by PR3 in primary microglia and striatum, which also prevented neuronal cell death in both conditions. The data presented here suggest that PR3 is a direct modulator of microglial activation and causes neuronal death through the augmentation of inflammatory responses. We suggest that PR3 could be a new modulator of neuroinflammation, and blocking PR3 would be a promising novel therapeutic target for neuroinflammatory disease such as stroke and Alzheimer's disease.

Background: Monitoring of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) in remission usually includes indirect immunofluorescence (IIF), proteinase 3 (PR3)-ANCA and myeloperoxidase (MPO)-ANCA. Typically, PR3 and MPO-ANCA are both performed because patients sometimes switch specificity during follow up. Published data are limited to case reports and incidence of change is not reported. The aim of this study was to quantify the incidence of antibody switching., Methods: Hull and East Yorkshire Hospitals National Health Service Trust serves a population of 720,000 for ANCA testing. We reviewed all ANCA results from January 2000 to August 2012 to quantify incidence of antibody switching. A total of 22,002 IIF screens (14,518 patients) were performed. A total of 9838 (45%) also had PR3- and MPO-ANCA (6439, 44% of patients). Patients that changed specificity from PR3- to MPO-ANCA and vice versa were identified and case notes reviewed., Results: A total of 218 patients with confirmed AAV positive for PR3/MPO-ANCA were followed for a mean of 2.6 years (range <0.1 to 12.4 years; with 113 (52%) patients followed for >1 year). Five patients (2%) changed specificity during follow up (3 GPA, 1 MPA & 1 EGPA). In two patients this was associated with relapse. Incidence of specificity change was 1 per 82 years (including two reversions to presenting specificity) and one per 286 years for changes associated with relapse. Monitoring using only the initial antibody specificity would have resulted in missed relapse in one patient., Conclusion: Antibody specificity changes in AAV are rare. Monitoring only the initial antibody specificity would have missed clinical events but rising C-reactive protein presaged relapse in these cases., (© The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.)

Proteinase 3 (Pr3), and human neutrophil elastase (HNE) are two major neutrophilic serine proteases (NsPs) expressed in neutrophil azurophil granules. Emerging data suggest that excessive release of proteases mediates tissue damage, and therefore prolonged neutrophil accumulation has an important role in the pathogenesis of many diseases. Thus, HNE and Pr3 inhibitors may prove to be targets for the generation of agents in the treatment of neutrophilic inflammatory disease. Sivelestat is the only commercially available selective HNE inhibitor. Therefore, sivelestat was chosen as the model structure in an attempt to obtain more potent anti-NsPs agents. In the present study, a series 2-aminobenzaldehyde oxime and 2-aminobenzoate analogs were synthesized and their inhibitory effects on NsPs (CatG, Pr3, and HNE) were determined, respectively. The results of structure-activity relationships studies concluded that a hydroxyl oxime moiety plays an important role in ligand-enzyme affinity through hydrogen bonding. As compound 6 had more potency and showed dual inhibitory effects on NE and Pr3, both in vitro and in vivo experiments were carried out to evaluate its selectivity, effects in cell-based assays, and efficacy in models of inflammation and damage. Compound 6 had the potential to reduce paw edema induced by LPS and HNE, as well as acute lung injury, and may be approved as a candidate for the development of new agents in the treatment of neutrophilic inflammatory diseases., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Bacterial biofilms have emerged as potential critical triggers in the pathogenesis of bisphosphonate (BP)-related osteonecrosis of the jaw (ONJ) or BRONJ. BRONJ lesions have shown to be heavily colonized by oral bacteria, most of these difficult to cultivate and presents many clinical challenges. The purpose of this study was to characterize the bacterial diversity in BRONJ lesions and to determine host immune response. We examined tissue specimens from three cohorts (n=30); patients with periodontal disease without a history of BP therapy (Control, n=10), patients with periodontal disease having history of BP therapy but without ONJ (BP, n=5) and patients with BRONJ (BRONJ, n=15). Denaturing gradient gel electrophoresis of polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments revealed less bacterial diversity in BRONJ than BP and Control cohorts. Sequence analysis detected six phyla with predominant affiliation to Firmicutes in BRONJ (71.6%), BP (70.3%) and Control (59.1%). Significant differences (P<0.05) in genera were observed, between Control/BP, Control/BRONJ and BP/BRONJ cohorts. Enzyme-linked immunosorbent assay (ELISA) results indicated that the levels of myeloperoxidase were significantly lower, whereas interleukin-6 and tumor necrosis factor-alpha levels were moderately elevated in BRONJ patients as compared to Controls. PCR array showed significant changes in BRONJ patients with downregulation of host genes, such as nucleotide-binding oligomerization domain containing protein 2, and cathepsin G, the key modulators for antibacterial response and upregulation of secretory leukocyte protease inhibitor, proteinase 3 and conserved helix-loop-helix ubiquitous kinase. The results suggest that colonization of unique bacterial communities coupled with deficient innate immune response is likely to impact the pathogenesis of ONJ.

Neutrophil serine proteases, proteinase 3 (PR3) and human neutrophil elastase (HNE), are considered as targets for chronic inflammatory diseases. Despite sharing high sequence similarity, the two enzymes have different substrate specificities and functions. While a plethora of HNE inhibitors exist, PR3 specific inhibitors are still in their infancy. We have designed ketomethylene-based inhibitors for PR3 that show low micromolar IC50 values. Their synthesis was made possible by amending a previously reported synthesis of ketomethylene dipeptide isosteres to allow for the preparation of derivatives suitable for solid phase peptide synthesis. The best inhibitor (Abz-VADnV[Ψ](COCH2)ADYQ-EDDnp) was found to be selective for PR3 over HNE and to display a competitive and reversible inhibition mechanism. Molecular dynamics simulations show that the interactions between enzyme and ketomethylene-containing inhibitors are similar to those with the corresponding substrates. We also confirm that N- and C-terminal FRET groups are important for securing high inhibitory potency toward PR3.

The function of neutrophil protease 3 (PR3) is poorly understood despite of its role in autoimmune vasculitides and its possible involvement in cell apoptosis. This makes it different from its structural homologue neutrophil elastase (HNE). Endogenous inhibitors of human neutrophil serine proteases preferentially inhibit HNE and to a lesser extent, PR3. We constructed a single-residue mutant PR3 (I217R) to investigate the S4 subsite preferences of PR3 and HNE and used the best peptide substrate sequences to develop selective phosphonate inhibitors with the structure Ac-peptidyl(P)(O-C6H4-4-Cl)2. The combination of a prolyl residue at P4 and an aspartyl residue at P2 was totally selective for PR3. We then synthesized N-terminally biotinylated peptidyl phosphonates to identify the PR3 in complex biological samples. These inhibitors resisted proteolytic degradation and rapidly inactivated PR3 in biological fluids such as inflammatory lung secretions and the urine of patients with bladder cancer. One of these inhibitors revealed intracellular PR3 in permeabilized neutrophils and on the surface of activated cells. They hardly inhibited PR3 bound to the surface of stimulated neutrophils despite their low molecular mass, suggesting that the conformation and reactivity of membrane-bound PR3 is altered. This finding is relevant for autoantibody binding and the subsequent activation of neutrophils in granulomatosis with polyangiitis (formerly Wegener disease). These are the first inhibitors that can be used as probes to monitor, detect, and control PR3 activity in a variety of inflammatory diseases., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Inheritance of the F variant of alpha-1-antitrypsin is associated with normal circulating protein levels, but it is believed to be dysfunctional in its ability to inhibit neutrophil elastase and therefore has been implicated as a susceptibility factor for the development of emphysema. In this study, its functional characteristics were determined following the identification of a unique patient with the PiFF phenotype, and the implications as a susceptibility factor for emphysema are considered both in homozygotes and heterozygotes., Methods: Second order association rate constants were measured for M, Z, S and F variants of alpha-1-antitrypsin with neutrophil elastase and proteinase 3. Clinical characteristics of the PiFF homozygote and six PiFZ heterozygote subjects were studied., Results: The F variant had a reduced association rate constant with neutrophil elastase (5.60 ± 0.83 × 106 M-1 s-1) compared to the normal M variant (1.45 ± 0.02 × 107 M-1 s-1), indicating an increased time to inhibition that was comparable to that of the Z variant (7.34 ± 0.03 × 106 M-1 s-1). The association rate constant for the F variant and proteinase 3 (1.06 ± 0.22 × 106 M-1 s-1) was reduced compared to that with neutrophil elastase, but was similar to that of other alpha-1-antitrypsin variants. Of the six PiFZ heterozygotes, five had airflow obstruction and radiological evidence of emphysema. The PiFF homozygote had airflow obstruction but no emphysema. None of the patients had clinical evidence of liver disease., Conclusions: The F variant may increase susceptibility to elastase-induced lung damage but not emphysema, whereas co-inheritance with the Z deficiency allele may predispose to emphysema despite reasonable plasma concentrations of alpha-1-antitrypsin.

Microscopic Polyangiitis (MPA) is a small vessel vasculitis. The disease is defined by the 2012 revised Chapel Hill Consensus Conference Nomenclature of Vasculitides [1] as necrotizing vasculitis, with few or no immune deposits, predominantly affecting small vessels (i.e. capillaries, venules, or arterioles). Necrotizing arteritis involving small and medium arteries may be present. Necrotizing glomerulonephritis is very common. Pulmonary capillaritis often occurs. Granulomatous inflammation is absent. MPA belongs to the ANCA-associated vasculitides (AAV). ANCA in MPA are predominantly directed against myeloperoxidase (MPO-ANCA) but may, in a minority of patients, be directed against proteinase 3 (PR3-ANCA). Not all patients, however, have ANCA. Microscopic polyangiitis (MPA) belongs to the anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides. MPA is clinically characterized by small-vessel vasculitis primarily affecting the kidneys and the lungs but other organs may be involved as well. Renal involvement, which can be the only manifestation, is clinically apparent as rapidly progressive glomerulonephritis and histopathologically as pauci-immune necrotizing and crescentic glomerulonephritis. ANCA in MPA are mainly directed to myeloperoxidase (MPO-ANCA). Besides their diagnostic significance, MPO-ANCA appear pathogenic in MPA. Rituximab with steroids is at least as effective as cyclophosphamide with steroids for induction of remission., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Neutrophils are among the first cells implicated in acute inflammation. Leaving the blood circulation, they quickly migrate through the interstitial space of tissues and liberate oxidants and other antimicrobial proteins together with serine proteinases. Neutrophil elastase, cathepsin G, proteinase 3 (PR3), and neutrophil serine protease 4 are four hematopoietic serine proteases activated by dipeptidyl peptidase I during neutrophil maturation and are mainly stored in cytoplasmic azurophilic granules. They regulate inflammatory and immune responses after their release from activated neutrophils at inflammatory sites. Membrane-bound PR3 (mbPR3) at the neutrophil surface is the prime antigenic target of antineutrophil cytoplasmic autoantibodies (ANCA) in granulomatosis with polyangiitis (GPA), a vasculitis of small blood vessels and granulomatous inflammation of the upper and/or lower respiratory tracts. The interaction of ANCA with mbPR3 results in excessive activation of neutrophils to produce reactive oxygen species and liberation of granular proteinases to the pericellular environment. In this review, we focus on PR3 and dipeptidyl peptidase I as attractive pharmacological targets whose inhibition is expected to attenuate autoimmune activation of neutrophils in GPA.

C5a and the neutrophil C5a receptor play a central role in antineutrophil cytoplasmic antibody (ANCA)-mediated neutrophil recruitment and activation. Our recent study found that activation of p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase (ERK) and phosphoinositol 3-kinase (PI3K) are all important steps in the translocation of ANCA antigens by C5a-mediated priming and activation of neutrophils. The current study further investigated the protein kinase C (PKC) pathway of C5a-mediated neutrophils for ANCA-induced activation. The effect of the PKC inhibitor (bisindolylmaleimide I, BIS) was tested on respiratory burst and degranulation of C5a-primed neutrophils activated with ANCA, as well as on C5a-induced increase in expression of PR3 and MPO. For C5a-primed neutrophils for MPO-ANCA-induced respiratory burst, the mean fluorescence intensity (MFI) value was 369.8±18.8, which decreased to 308.3±14.2 upon pre-incubation with BIS (P<0.001). For PR3-ANCA-positive IgG, the MFI value increased in C5a-primed neutrophils, which decreased upon pre-incubation with BIS. The lactoferrin concentration increased from 414.8±26.9 ng/ml in the non-primed neutrophils supernatant to 1099.8±80.7 ng/ml in C5a-primed neutrophils induced by MPO-ANCA-positive IgG supernatant (P<0.001), and decreased to 814.5±45.3 ng/ml upon pre-incubation with BIS (P<0.01). The lactoferrin concentration also increased in C5a-primed neutrophils induced by PR3-ANCA-positive IgG supernatant and decreased upon pre-incubation with BIS. Membrane expression of PR3 (mPR3) expression increased from 788.0±87.5 in untreated cells to 1071.3±81.3 after C5a treatment and decreased to 827.3±48.1 by BIS (P<0.05). Activation of PKC is an important step in the translocation of ANCA antigens and C5a-induced activation of neutrophils by ANCA., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Human neutrophil proteinase 3 (PR3) and elastase (HNE) are homologous serine proteinases involved in the proteolytic events associated with inflammation and infection. Their close structural and functional resemblance makes it difficult to understand their respective biological functions. Thus, all natural inhibitors of PR3 identified to date preferentially target HNE, and only recently have inhibitors that target PR3 selectively been described. This review describes how differences in the structures of the extended active sites of PR3 and HNE can be exploited to produce selective inhibitors of PR3.

A series of bi- and tricyclic β-lactam compounds was synthesized and evaluated as inhibitors of cleavage of synthetic substrates in vitro by the serine proteases Human Leukocyte Elastase (HLE), Human Leukocyte Proteinase 3 (HLPR3) and Porcine Pancreatic Elastase (PPE). The obtained results have permitted us to describe a homobenzocarbacephem compound as HLE and HLPR3 inhibitor, to observe the positive effect that the styryl group exerts on the HLE inhibitory activity in polycyclic β-lactam compounds and to conclude that the hydroxyl function decreases the HLE inhibitory activity or rules it out completely., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The biological functions of human neutrophil proteinase 3 (PR3) remain unclear because of its close structural resemblance to neutrophil elastase and its apparent functional redundancy with the latter. Thus, all natural inhibitors of PR3 preferentially target neutrophil elastase. We have designed a selective PR3 inhibitor based on the sequence of one of its specific, sensitive FRET substrates. This azapeptide, azapro-3, inhibits free PR3 in solution, PR3 bound to neutrophil membranes, and the PR3 found in crude lung secretions from patients with chronic inflammatory pulmonary diseases. But it does not inhibit significantly neutrophil elastase or cathepsin G. Unlike most of azapeptides, this inhibitor does not form a stable acyl-enzyme complex; it is a reversible competitive inhibitor with a K(i) comparable to the K(m) of the parent substrate. Low concentrations (60 μM) of azapro-3 totally inhibited the PR3 secreted by triggered human neutrophils (200,000 cells/100 μL) and the PR3 in neutrophil homogenates and in lung secretions of patients with lung inflammation for hours. Azapro-3 also resisted proteolysis by all proteases contained in these samples for at least 2h., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The physiological and pathological functions of proteinase 3 (PR3) are not well understood due to its close similarity to human neutrophil elastase (HNE) and the lack of a specific inhibitor. Based on structural analysis of the active sites of PR3 and HNE, we generated mutants derived from the polyvalent inhibitor SerpinB1 (monocyte/neutrophil elastase inhibitor) that specifically inhibit PR3 and that differ from wt-SerpinB1 by only 3 or 4 residues in the reactive center loop. The rate constant of association between the best SerpinB1 mutant and PR3 is 1.4 × 10⁷ M⁻¹ · s⁻¹, which is ∼100-fold higher than that observed with wt-SerpinB1 and compares with that of α1-protease inhibitor (α1-PI) toward HNE. SerpinB1(S/DAR) is cleaved by HNE, but due to differences in rate, inhibition of PR3 by SerpinB1(S/DAR) is only minimally affected by the presence of HNE even when the latter is in excess. SerpinB1(S/DAR) inhibits soluble PR3 and also membrane-bound PR3 at the surface of activated neutrophils. Moreover, SerpinB1(S/DAR) clears induced PR3 from the surface of activated neutrophils. Overall, these specific inhibitors of PR3 will be valuable for defining biological functions of the protease and may prove useful as therapeutics for PR3-related inflammatory diseases, such as Wegener's granulomatosis.

Polymorphonuclear neutrophils are the first cells recruited to inflammatory sites and form the earliest line of defense against invading microorganisms. Neutrophil elastase, proteinase 3, and cathepsin G are three hematopoietic serine proteases stored in large quantities in neutrophil cytoplasmic azurophilic granules. They act in combination with reactive oxygen species to help degrade engulfed microorganisms inside phagolysosomes. These proteases are also externalized in an active form during neutrophil activation at inflammatory sites, thus contributing to the regulation of inflammatory and immune responses. As multifunctional proteases, they also play a regulatory role in noninfectious inflammatory diseases. Mutations in the ELA2/ELANE gene, encoding neutrophil elastase, are the cause of human congenital neutropenia. Neutrophil membrane-bound proteinase 3 serves as an autoantigen in Wegener granulomatosis, a systemic autoimmune vasculitis. All three proteases are affected by mutations of the gene (CTSC) encoding dipeptidyl peptidase I, a protease required for activation of their proform before storage in cytoplasmic granules. Mutations of CTSC cause Papillon-Lefèvre syndrome. Because of their roles in host defense and disease, elastase, proteinase 3, and cathepsin G are of interest as potential therapeutic targets. In this review, we describe the physicochemical functions of these proteases, toward a goal of better delineating their role in human diseases and identifying new therapeutic strategies based on the modulation of their bioavailability and activity. We also describe how nonhuman primate experimental models could assist with testing the efficacy of proposed therapeutic strategies.

Proteinase 3 (PR3)-specific antineutrophil cytoplasmic antibodies (ANCA) are highly specific for the autoimmune small vessel vasculitis, Wegener's granulomatosis (WG). PR3-ANCA have proven diagnostic value but their pathogenic potential and utility as a biomarker for disease activity remain unclear. PR3-ANCA recognize conformational epitopes, and epitope-specific PR3-ANCA subsets with variable impact on biological functions of PR3 have been postulated. The aims of this study were to identify specific PR3 surface epitopes recognized by monoclonal antibodies (moAbs) and to determine whether the findings can be used to measure the functional impact of epitope-specific PR3-ANCA and their potential relationship to disease activity. We used a novel flow cytometry assay based on TALON-beads coated with recombinant human (H) and murine (M) PR3 and 10 custom-designed chimeric human/mouse rPR3-variants (Hm1-5/Mh1-5) identifying 5 separate non-conserved PR3 surface epitopes. Anti-PR3 moAbs recognize 4 major surface epitopes, and we identified the specific surface location of 3 of these with the chimeric rPR3-variants. The ability of PR3-ANCA to inhibit the enzymatic activity of PR3 was measured indirectly using a capture-ELISA system based on the different epitopes recognized by capturing moAbs. Epitope-specific PR3-ANCA capture-ELISA results obtained from patient plasma (n=27) correlated with the inhibition of enzymatic activity of PR3 by paired IgG preparations (r=0.7, P<0.01). The capture-ELISA results also seem to reflect disease activity. In conclusion, insights about epitopes recognized by anti-PR3 moAbs can be applied to separate PR3-ANCA subsets with predictable functional qualities. The ability of PR3-ANCA to inhibit the enzymatic activity of PR3, a property linked to disease activity, can now be gauged using a simple epitope-based capture-ELISA system., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

A structurally-diverse series of carboxylate derivatives based on the 1,2,5-thiadiazolidin-one 1,1 dioxide scaffold were synthesized and used to probe the S' subsites of human neutrophil elastase (HNE) and neutrophil proteinase 3 (Pr 3). Several compounds are potent inhibitors of HNE but devoid of inhibitory activity toward Pr 3, suggesting that the S' subsites of HNE exhibit significant plasticity and can, unlike Pr 3, tolerate various large hydrophobic groups. The results provide a promising framework for the design of highly selective inhibitors of the two enzymes., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

The S' subsites of human neutrophil proteinase 3 (Pr 3) were probed by constructing diverse libraries of compounds based on the 1,2,3,5-thiatriazolidin-3-one 1,1-dioxide using combinational and click chemistry methods. The multiple points of diversity embodied in the heterocyclic scaffold render it well-suited to the exploration of the S' subsites of Pr 3. Molecular modeling studies suggest that further exploration of the S' subsites of Pr 3 using the aforementioned heterocyclic scaffold may lead to the identification of highly selective, reversible competitive inhibitors of Pr 3., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

Proteinase 3 (Pr3), the main target of anti-neutrophil cytoplasmic antibodies, is a neutrophil serine protease that may be constitutively expressed at the surface of quiescent circulating neutrophils. This raises the question of the simultaneous presence in the circulation of constitutive membrane-bound Pr3 (mPr3) and its plasma inhibitor alpha1-protease inhibitor (alpha1-Pi). We have looked at the fate of constitutive mPr3 at the surface of circulating blood neutrophils and of induced mPr3 on triggered neutrophils. We found significant Pr3 activity at the surface of activated neutrophils but not at the surface of quiescent neutrophils whatever the constitutive expression. This suggests that constitutive mPr3 is enzymatically inactive or its active site is not accessible to the substrate. Supporting this conclusion, we have not been able to demonstrate any interaction between constitutive mPr3 and alpha1-Pi, whereas induced mPr3 is cleared from the cell surface when activated cells are incubated with this inhibitor. But, unlike membrane-bound elastase that is also cleared from the surface of activated cells, mPr3 remained bound to the membrane when inhibited by elafin or by a low molecular weight chloromethyl ketone inhibitor, which shows that it binds more tightly to the neutrophil membrane. mPr3 may thus be present at the surface of circulating neutrophils in an environment replete with alpha1-Pi. The permanent presence of inactive Pr3 at the surface of quiescent neutrophils may explain why Pr3 is a major target of anti-neutrophil cytoplasmic antibodies, whose binding activates neutrophils and triggers inflammation, as in Wegener granulomatosis.

A series of mechanism-based inhibitors designed to interact with the S' subsites of serine proteases was synthesized and their inhibitory activity toward the closely-related serine proteases human neutrophil elastase (HNE) and proteinase 3 (PR 3) was investigated. The compounds were found to be time-dependent inhibitors of HNE and were devoid of any inhibitory activity toward PR 3. The results suggest that highly selective inhibitors of serine proteases whose primary substrate specificity and active sites are similar can be identified by exploiting differences in their S' subsites. The best inhibitor (compound 16) had a k(inact)/K(I) value of 4580 M(-1)s(-1).

Eight new cyanopeptolins (insulapeptolides A-H) were obtained from the cyanobacterium Nostoc insulare. Their isolation was guided by their bioactivity toward the target enzyme human leukocyte elastase, molecular biological investigations, and MALDI-TOF analysis. These peptides are selective inhibitors of human leukocyte elastase with activities in the nanomolar range. Insulapeptolide D was the most potent compound with an IC(50) value of 85 nM (K(i) value of 36 nM).

Proteinase 3 (PR3) expression on neutrophils was examined in rheumatoid arthritis (RA) patients before and after antitumor necrosis factor (TNF)-alpha therapy. Membrane PR3 expression from patients with either an infection or RA significantly increased. Membrane PR3 expression on neutrophils from RA patients treated with infliximab (anti-TNF-alpha antibody) therapy was less than in those without such treatment in a resting state, but the expression later increased after stimulation in vitro. Membrane PR3 expression increased because of the stimulation of TNFalpha, whereas it was significantly suppressed by plasma or alpha(1)-proteinase inhibitor. The condition of patients with RA improved after treatment with infliximab. Membrane PR3 expression on neutrophils in RA patients was downregulated by infliximab. As a result, PR3 might play an important role in the neutrophil-mediated inflammatory reaction in patients with either RA or an infection.

The interaction of a series of 1,2,5-thiadiazolidin-3-one 1,1 dioxide-based sulfonamides with neutrophil-derived serine proteases was investigated. The nature of the amino acid component, believed to be oriented toward the S' subsites, had a profound effect on enzyme selectivity. This series of compounds were found to be potent, time-dependent inhibitors of human neutrophil elastase (HNE) and were devoid of any inhibitory activity toward neutrophil proteinase 3 (PR 3) and cathepsin G (Cat G). The results of these studies demonstrate that exploitation of differences in the S' subsites of HNE and PR 3 can lead to highly selective inhibitors of HNE.

Anti-proteinase-3 (anti-PR3) or anti-myeloperoxidase (anti-MPO) antibodies are capable of activating human neutrophils primed by TNF-alpha in vitro. We described previously the involvement of FcgammaRIIa and beta(2) integrins in this neutrophil activation. In the literature, the requirement of TNF priming has been attributed to an effect of TNF-alpha on the expression of PR3 or MPO on the cell surface. Under our experimental conditions, TNF-alpha (2 ng/ml) increased the binding of the antibody against PR3, whereas binding of the antibody against MPO could hardly be detected, not even after TNF-alpha treatment. The aim of this study was to consider (an)other(s) role(s) for TNF-alpha in facilitating the NADPH-oxidase activation by these antibodies. We demonstrate the early mobilization of the secretory vesicles as a result of TNF-induced increase in intracellular-free calcium ions, the parallel colocalization of gp91(phox), the main component of the NADPH oxidase with beta(2) integrins and FcgammaRIIa on the neutrophil surface, and the FcgammaRIIa clustering upon TNF priming. TNF-alpha also induced redistribution of FcgammaRIIa to the cytoskeleton in a dose- and time-dependent manner. Moreover, blocking CD18 MHM23 antibody, cytochalasin B, and D609 (an inhibitor of phosphatidylcholine phospholipase C) inhibited this redistribution and the respiratory burst in TNF-treated neutrophils exposed to anti-PR3 or anti-MPO antibodies. Our results indicate direct effects of TNF-alpha in facilitating neutrophil activation by these antibodies and further support the importance of cytoskeletal rearrangements in this priming process.