Cytosolic proliferating cell nuclear antigen (PCNA) binds to procaspases and protects human neutrophils from apoptosis.

No abstract

Annexin 1 is an anti-inflammatory protein that plays a key role in innate immunity by modulating the activation of several types of cells, including neutrophils. Here we have developed a cleavage assay using tagged annexin 1 and observed marked activity in the membrane fraction of activated neutrophils. A combination of inhibitors, transfected cells, and proteomic analyses allowed us to identify proteinase 3 as the main enzyme responsible for this cleavage in the N terminus region of the protein, at least in the context of neutrophil activation. Because annexin 1 is an important endogenous anti-inflammatory mediator, blocking its cleavage by proteinase 3 would augment its homeostatic pro-resolving actions and could represent an opportunity for innovative anti-inflammatory drug discovery.

Proteinase 3 (PR3), a serine proteinase contained in neutrophil azurophilic granules, is considered a risk factor for vasculitides and rheumatoid arthritis when expressed on the outer leaflet of neutrophil plasma membrane and is the preferred target of antineutrophil cytoplasm autoantibodies (ANCA) in Wegener granulomatosis. ANCA binding to PR3 expressed at the surface of neutrophils activates them. Evidence is provided that neutrophil apoptosis induced significantly more membrane PR3 expression without degranulation (but no enhanced membrane CD35, IntroductionProteinase 3 (PR3), also called myeloblastin, 1 belongs to the family of neutrophil microbicidal serine proteinases that are stored within azurophilic granules. 2 They are considered proinflammatory proteinases because they mediate deleterious effects on host tissues during inflammation. 3,4 Intriguingly, among the numerous proteins contained within azurophilic granules, only PR3 and myeloperoxidase (MPO) are the main targets of antineutrophil cytoplasm antibodies (ANCA) associated with systemic vasculitides. 5,6 More than 80% of Wegener granulomatosis patients have anti-PR3 ANCA, whereas only 10% have anti-MPO ANCA. By contrast, microscopic polyangiitis, Churg-Strauss syndrome, and pauci-immune crescentic glomerulosclerosis are generally associated with anti-MPO ANCA. 7 Unlike MPO, whose subcellular localization is restricted to azurophilic granules, PR3 has been detected on secretory vesicle membranes and the outer leaflet of plasma membranes. 8,9 ANCA have a pathophysiologic role because they activate neutrophils when ANCA antigens are expressed at the neutrophil membrane. 7 We and others demonstrated that high percentages of membrane PR3-positive neutrophils could favor the development or progression of chronic inflammatory diseases, namely vasculitides and rheumatoid arthritis. [10][11][12] Moreover, enhanced membrane PR3 expression was observed on neutrophils from patients with sepsis. 13 Taken together, these data strongly suggest that membrane PR3 expression constitutes a pathogenic factor in ANCA-associated vasculitis and other inflammatory diseases involving neutrophils.Because of the unexpected complex subcellular localization of PR3 in neutrophils, we previously investigated whether it could have specific molecular substrates and, consequently, unanticipated functions. To explore this possibility, we used the rat mast cell lines (RBL-2H3) stably transfected with human neutrophil elastase (HNE), PR3, or its inactive mutant PR3S203A and demonstrated that PR3, unlike its homolog HNE, was able to access cytosolic (eg, the cyclin-dependent kinase inhibitor p21/waf1) 14 or membrane substrates (eg, procaspase-3). 15 We also observed that PR3 could be expressed at the cell surface during apoptosis, independently of degranulation, and was strongly associated with phosphatidylserine (PS) externalization, 16 which is an "eat-me" signal recognized by macrophages. 17,18 Thus, it became evident, at least in our model of stable transfectants, that PR3 was lo...

Polymorphonuclear neutrophils (PMNs) and monocyte/macrophages (MMs) are professional phagocytic cells that are able to phagocytose and destroy infectious agents. Therefore, they are key anti-infectious actors in host defense but can mediate tissue damages. In addition, it is now clear that the role of these cells goes far beyond phagocytosis and pathogen killing. PMNs and MMs are essential cells for immunity, absolutely required to build and modulate the innate response. The respective roles of PMNs and MMs in the inflammatory process are discussed: their common features and their differences are reviewed, both in terms of origins and functions with special emphasis on novel concepts about neutrophil survival and resolution of inflammation. The recognition and the subsequent engulfment of apoptotic PMNs by macrophages is a key event of the resolution of inflammation, which can be associated with autoimmunity or inflammatory diseases. During the past years, significant efforts have been made to dissect the molecular mechanisms governing phagocytosis and pathogen killing. Although these effector functions are crucial, more work has to be done to understand the respective role of PMNs and MMs to regulate and inhibit the inflammatory process as well as the immune response. This might be the future challenge for the next years in phagocyte research and this will presumably open new avenues of research in the modulation of inflammation.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides are a heterogeneous group of diseases corresponding to necrotising inflammation of small vessels with a wide range of clinical presentations. At least two of the diseases are believed to exhibit a common ground of pathophysiological mechanisms. These are granulomatosis with polyangiitis (GPA, formerly known as Wegener's granulomatosis) and microscopic polyangiitis (MPA). ANCA directed against proteinase 3 (PR3) are preferentially associated with GPA, and anti-myeloperoxidase (MPO) ANCA are associated mainly with MPA and eosinophilic GPA (formerly known as Churg-Strauss syndrome). Anti-MPO and anti-PR3 antibodies can activate neutrophils in vitro. In vivo data are available for humans and mice on the pathogenicity of anti-MPO but it is more controversial for PR3-ANCA. A recent genome-wide association study of patients with ANCA-associated vasculitides confirmed the genetic contribution to the pathogenesis of these conditions, with significant association of PR3-ANCA and human leukocyte antigen-DP and the genes encoding α1-antitrypsin and PR3. MPO-ANCA were significantly associated with human leukocyte antigen-DQ. Thus, recent results from epidemiological studies, genome-wide association study and therapeutic trials have suggested that these entities are, in fact, distinct. We have summarised these results and discuss the idea that these two entities should be studied separately as the nature of the two auto-antigens suggests at a molecular level despite shared ANCA involvement.

Proteinase 3 (PR3) is the target of anti-neutrophil cytoplasm Abs in granulomatosis with polyangiitis, a form of systemic vasculitis. Upon neutrophil apoptosis, PR3 is coexternalized with phosphatidylserine and impaired macrophage phagocytosis. Calreticulin (CRT), a protein involved in apoptotic cell recognition, was found to be a new PR3 partner coexpressed with PR3 on the neutrophil plasma membrane during apoptosis, but not after degranulation. The association between PR3 and CRT was demonstrated in neutrophils by confocal microscopy and coimmunoprecipitation. Evidence for a direct interaction between PR3 and the globular domain of CRT, but not with its P domain, was provided by surface plasmon resonance spectroscopy. Phagocytosis of apoptotic neutrophils from healthy donors was decreased after blocking lipoprotein receptor-related protein (LRP), a CRT receptor on macrophages. In contrast, neutrophils from patients with granulomatosis with polyangiitis expressing high membrane PR3 levels showed a lower rate of phagocytosis than those from healthy controls not affected by anti-LRP, suggesting that the LRP-CRT pathway was disturbed by PR3-CRT association. Moreover, phagocytosis of apoptotic PR3-expressing cells potentiated proinflammatory cytokine in vitro by human monocyte-derived macrophages and in vivo by resident murine peritoneal macrophages, and diverted the anti-inflammatory response triggered by the phagocytosis of apoptotic cells after LPS challenge in thioglycolate-elicited murine macrophages. Therefore, membrane PR3 expressed on apoptotic neutrophils might amplify inflammation and promote autoimmunity by affecting the anti-inflammatory “reprogramming” of macrophages.

Human polymorphonuclear leukocytes adhesion to endothelial cells during the early stage of inflammation leads to cell surface externalization of Annexin A1 (AnxA1), an effector of endogenous anti-inflammation. The antiadhesive properties of AnxA1 become operative to finely tune polymorphonuclear leukocytes transmigration to the site of inflammation. Membrane bound proteinase 3 (PR3) plays a key role in this microenvironment by cleaving the N terminus bioactive domain of AnxA1. In the present study, we generated a PR3-resistant human recombinant AnxA1-named superAnxA1 (SAnxA1)-and tested its in vitro and in vivo properties in comparison to the parental protein. SAnxA1 bound and activated formyl peptide receptor 2 in a similar way as the parental protein, while showing a resistance to cleavage by recombinant PR3. SAnxA1 retained anti-inflammatory activities in the murine inflamed microcirculation (leukocyte adhesion being the readout) and in skin trafficking model. When longer-lasting models of inflammation were applied, SAnxA1 displayed stronger anti-inflammatory effect over time compared with the parental protein. Together these results indicate that AnxA1 cleavage is an important process during neutrophilic inflammation and that controlling the balance between AnxA1/PR3 activities might represent a promising avenue for the discovery of novel therapeutic approaches