ABSTRACT
Development of very-low field MRI is an active area of research. It aims at reducing operating costs and improve portability. However, the signal-to-noise issue becomes prominent at ultra-low field (<1 mT), especially for molecular imaging purposes that addresses specific biochemical events. In the context of preclinical molecular MRI of abnormal proteolysis the paper describes a MRI system able to produce Overhauser-enhanced MR images in living rats through in situ Dynamic Nuclear Polarization at 206 µT using stable and non-toxic nitroxides. In parallel conventional images are generated at 206 µT following pre-polarization at 20 mT. Results show that nitroxides are visualized in 3D within a few minutes in the lungs, kidneys and bladder post-administration. This system will be used for molecular imaging of inflammation using protease-specific nitroxide probes.
Subject(s)
Lung , Magnetic Resonance Imaging , Rats , Animals , Magnetic Resonance Spectroscopy , Magnetic Resonance Imaging/methods , Nitrogen Oxides/chemistryABSTRACT
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 TNFα-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 antigen-binding 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.
Subject(s)
Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis , Antibodies, Antineutrophil Cytoplasmic , Myeloblastin , Animals , Humans , Mice , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/metabolism , Myeloblastin/immunology , Myeloblastin/metabolism , Neutrophils/metabolism , Protease Inhibitors/metabolism , Superoxides/metabolismABSTRACT
Current chemotherapies suffer low specificity and sometimes drug resistance. Neutrophil elastase activity in cancer is associated with poor prognosis and metastasis settlement. More generally, tumors harbor various and persistent protease activities unseen in healthy tissues. In an attempt to be more specific, we designed prodrugs that are activatable by neutrophil elastase. Upon activation, these alkoxyamine-based drugs release cytotoxic alkyl radicals that act randomly to prevent drug resistance. As a result, U87 glioblastoma cells displayed high level caspase 3/7 activation during the first hour of exposure in the presence of human neutrophil elastase and the prodrug in vitro. The apoptosis process and cell death occurred between 24 and 48 h after exposure with a half lethal concentration of 150 µM. These prodrugs are versatile and easy to synthetize and can be adapted to many enzymes.
Subject(s)
Antineoplastic Agents , Glioblastoma , Prodrugs , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Glioblastoma/pathology , Humans , Leukocyte Elastase/metabolism , Neutrophils/metabolism , Prodrugs/metabolismABSTRACT
A near-infrared fluorescent (NIRF) substrate-based probe (SBP) was conceived to monitor secreted human proteinase 3 (hPR3) activity. This probe, called pro3-SBP, is shaped by a fused peptide hairpin loop structure, which associates a hPR3 recognition domain (Val-Ala-Asp-Nva-Ala-Asp-Tyr-Gln, where Nva is norvaline) and an electrostatic zipper (consisting of complementary polyanionic (d-Glu)5 and polycationic (d-Arg)5 sequences) in close vicinity of the N- and C-terminal FRET couple (fluorescent donor, sulfoCy5.5; dark quencher, QSY21). Besides its subsequent stability, no intermolecular fluorescence quenching was detected following its complete hydrolysis by hPR3, advocating that pro3-SBP could further afford unbiased imaging. Pro3-SBP was specifically hydrolyzed by hPR3 (kcat/Km= 440â¯000 ± 5500 M-1·s-1) and displayed a sensitive detection threshold for hPR3 (subnanomolar concentration range), while neutrophil elastase showed a weaker potency. Conversely, pro3-SBP was not cleaved by cathepsin G. Pro3-SBP was successfully hydrolyzed by conditioned media of activated human neutrophils but not by quiescent neutrophils. Moreover, unlike unstimulated neutrophils, a strong NIRF signal was specifically detected by confocal microscopy following neutrophil ionomycin-induced degranulation. Fluorescence release was abolished in the presence of a selective hPR3 inhibitor, indicating that pro3-SBP is selectively cleaved by extracellular hPR3. Taken together, the present data support that pro3-SBP could be a convenient tool, allowing straightforward monitoring of human neutrophil activation.
Subject(s)
Myeloblastin/metabolism , Neutrophil Activation/physiology , Neutrophils/physiology , Cell Survival , Fluorescent Dyes , Gene Expression Regulation, Enzymologic/drug effects , Humans , Ionomycin , Microscopy, Confocal , Molecular Structure , Myeloblastin/chemistry , Neutrophils/drug effects , Spectrophotometry, InfraredSubject(s)
COVID-19 , Humans , Neutrophils , Peptide Hydrolases , Respiration, Artificial , SARS-CoV-2ABSTRACT
Granulomatosis with polyangiitis (GPA) is a severe autoimmune vasculitis associated with the presence of anti-neutrophil cytoplasmic antibodies (ANCA) mainly targeting proteinase 3 (PR3), a neutrophilic serine proteinase. PR3-ANCA binding to membrane-bound PR3 on neutrophils induce their auto-immune activation responsible for vascular lesions. However, the correlation between PR3-ANCA level and disease activity remains inconsistent, suggesting the existence of non-pathogenic PR3-ANCA. In order to prove their existence, we immortalized B lymphocytes from blood samples of GPA patients in remission having persistent PR3-ANCA to isolate non-activating PR3-ANCA. We obtained for the first time a non-activating human IgG1κ anti-PR3 monoclonal antibody (mAb) named 4C3. This new mAb binds soluble PR3 with a high affinity and membrane-bound PR3 on an epitope close to the PR3 hydrophobic patch and in the vicinity of the active site. 4C3 is able to bind FcγRIIA and FcγRIIIB and has a G2F glycosylation profile on asparagine 297. 4C3 did not induce activation of neutrophils and could inhibit human polyclonal PR3-ANCA-induced activation suggesting that 4C3 is non-pathogenic. This characteristic relies on the recognized epitope on PR3 rather than to the Fc portion properties. The existence of non-pathogenic PR3-ANCA, which do not activate neutrophils, could explain the persistence of high PR3-ANCA levels in some GPA patients in remission and why PR3-ANCA would not predict relapse. Finally, these results offer promising perspectives particularly regarding the understanding of PR3-ANCA pathogenicity and the development of new diagnostic and therapeutic strategies in GPA.
Subject(s)
Antibodies, Antineutrophil Cytoplasmic/immunology , Antibodies, Monoclonal/immunology , B-Lymphocytes/immunology , Granulomatosis with Polyangiitis/immunology , Myeloblastin/immunology , Aged , Antibodies, Antineutrophil Cytoplasmic/metabolism , Antibodies, Monoclonal/metabolism , Antibody Affinity , Antibody Specificity , B-Lymphocytes/enzymology , Binding Sites, Antibody , Biomarkers/metabolism , Case-Control Studies , Cell Line , Epitope Mapping , Epitopes , Female , Glycosylation , Granulomatosis with Polyangiitis/diagnosis , Granulomatosis with Polyangiitis/enzymology , Humans , Male , Middle Aged , Neutrophil Activation , Proof of Concept StudyABSTRACT
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.
Subject(s)
Molecular Docking Simulation , Myeloblastin/chemistry , Pregnancy-Associated alpha 2-Macroglobulins/chemistry , Recombinant Proteins/chemistry , Amino Acid Sequence , Binding Sites , Chromatography, Liquid/methods , Humans , Kinetics , Mass Spectrometry/methods , Myeloblastin/genetics , Myeloblastin/metabolism , Peptides/chemistry , Peptides/genetics , Peptides/metabolism , Pregnancy-Associated alpha 2-Macroglobulins/genetics , Pregnancy-Associated alpha 2-Macroglobulins/metabolism , Protein Binding , Protein Domains , Proteolysis , Recombinant Proteins/metabolismABSTRACT
Cysteine cathepsin C (CatC) is a ubiquitously expressed, lysosomal aminopeptidase involved in the activation of zymogens of immune-cell-associated serine proteinases (elastase, cathepsin G, proteinase 3, neutrophil serine proteinase 4, lymphocyte granzymes, and mast cell chymases). CatC is first synthetized as an inactive zymogen containing an intramolecular chain propeptide, the dimeric form of which is processed into the mature tetrameric form by proteolytic cleavages. A molecular modeling analysis of proCatC indicated that its propeptide displayed a similar fold to those of other lysosomal cysteine cathepsins, and could be involved in dimer formation. Our in vitro experiments revealed that human proCatC was processed and activated by CatF, CatK, and CatV in two consecutive steps of maturation, as reported for CatL and CatS previously. The unique positioning of the propeptide domains in the proCatC dimer complex allows this order of cleavages to be understood. The missense mutation Leu172Pro within the propeptide region associated with the Papillon-Lefèvre and Haim-Munk syndrome altered the proform stability as well as the maturation of the recombinant Leu172Pro proform.
Subject(s)
Cathepsin C/chemistry , Enzyme Precursors/chemistry , Models, Molecular , Molecular Conformation , Binding Sites , Humans , Protein Binding , Recombinant Proteins/chemistryABSTRACT
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.
Subject(s)
Cathepsin C/antagonists & inhibitors , Cell Membrane/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Granulomatosis with Polyangiitis/pathology , Myeloblastin/metabolism , Adolescent , Adult , Case-Control Studies , Child , Child, Preschool , Female , Granulomatosis with Polyangiitis/drug therapy , Granulomatosis with Polyangiitis/genetics , Granulomatosis with Polyangiitis/metabolism , Humans , Male , Myeloblastin/genetics , Neutrophils/enzymology , Proteolysis , Young AdultABSTRACT
Cathepsin C (CatC) is a highly conserved tetrameric lysosomal cysteine dipeptidyl aminopeptidase. The best characterized physiological function of CatC is the activation of pro-inflammatory granule-associated serine proteases. These proteases are synthesized as inactive zymogens containing an N-terminal pro-dipeptide, which maintains the zymogen in its inactive conformation and prevents premature activation, which is potentially toxic to the cell. The activation of serine protease zymogens occurs through cleavage of the N-terminal dipeptide by CatC during cell maturation in the bone marrow. In vivo data suggest that pharmacological inhibition of pro-inflammatory serine proteases would suppress or attenuate deleterious effects mediated by these proteases in inflammatory/auto-immune disorders. The pathological deficiency in CatC is associated with Papillon-Lefèvre syndrome (PLS). The patients however do not present marked immunodeficiency despite the absence of active serine proteases in immune defense cells. Hence, the transitory pharmacological blockade of CatC activity in the precursor cells of the bone marrow may represent an attractive therapeutic strategy to regulate activity of serine proteases in inflammatory and immunologic conditions. A variety of CatC inhibitors have been developed both by pharmaceutical companies and academic investigators, some of which are currently being employed and evaluated in preclinical/clinical trials.