Mitochondria play a central role in maintaining cells alive, but are also important mediators of cell death. The main event in mitochondrial signalling and control of apoptosis is the permeabilisation of the outer mitochondrial membrane and the release of pro-apoptotic proteins into the cytosol from the mitochondrial intermembrane space. With respect to death receptor-mediated apoptosis, the activation of the mitochondrial pathway is required for apoptosis induction in cells which are described as "type II" cells whereas "type I" cells do not require it. In type I cells, activation of the extrinsic pathway is sufficient to induce apoptosis. This review deals with the events that enable cell death in type II cells, i.e., the signals that lead from death receptor stimulation to permeabilisation of the outer mitochondrial membrane. Caspase-8 and Bid are the known procurers of the death signal in this part of the apoptotic pathway. Currently many exciting new findings are emerging concerning the regulation of caspase-8 and Bid function and activation. We will take you on a journey through these new developments and point out what we consider the major unknowns in this field. We end our review on an up-to-date discussion of the determinants of the type I-type II cell distinction. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.

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.

Proteinase 3 and neutrophil elastase are serine proteinases of the polymorphonuclear neutrophils, which are considered to have both similar localization and ligand specificity because of their high sequence similarity. However, recent studies indicate that they might have different and yet complementary physiologic roles. Specifically, proteinase 3 has intracellular specific protein substrates resulting in its involvement in the regulation of intracellular functions such as proliferation or apoptosis. It behaves as a peripheral membrane protein and its membrane expression is a risk factor in chronic inflammatory diseases. Moreover, in contrast to human neutrophil elastase, proteinase 3 is the preferred target antigen in Wegener’s granulomatosis, a particular type of vasculitis. We review the structural basis for the different ligand specificities and membrane binding mechanisms of both enzymes, as well as the putative anti‐neutrophil cytoplasm autoantibody epitopes on human neutrophil elastase 3. We also address the differences existing between murine and human enzymes, and their consequences with respect to the development of animal models for the study of human proteinase 3‐related pathologies. By integrating the functional and the structural data, we assemble many pieces of a complicated puzzle to provide a new perspective on the structure–function relationship of human proteinase 3 and its interaction with membrane, partner proteins or cleavable substrates. Hence, precise and meticulous structural studies are essential tools for the rational design of specific proteinase 3 substrates or competitive ligands that modulate its activities.

Because neutrophil apoptosis plays a key role in resolving inflammation, identification of proteins regulating neutrophil survival should provide new strategies to modulate inflammation. Using a proteomic approach, coronin-1 was identified as a cytosolic protein cleaved during neutrophil apoptosis. Coronin-1 is an actin-binding protein that can associate with phagosomes and NADPH oxidase, but its involvement in apoptosis was currently unknown. In coronin-1-transfected PLB985 cells, coronin-1 overexpression did not modify the kinetics of granulocyte differentiation as assessed by CD11b labeling. Concerning apoptosis, increased coronin-1 expression in dimethylformamide-differentiated PLB985 significantly decreased gliotoxin-induced mitochondrial depolarization as compared with controls. Likewise, coronin-1 significantly decreased TRAIL-induced apoptosis with less mitochondrial depolarization, caspase-3 and caspase-9 activities, but not caspase-8 or Bid truncation suggesting that coronin-1 interfered with mitochondria-related events. To validate the prosurvival role of coronin-1 in a pathophysiological condition involving neutrophil-dominated inflammation, neutrophils from cystic fibrosis (CF) patients were studied. Circulating neutrophils from CF patients had more coronin-1 expression assessed by immunoblotting or proteomic analysis of cytosolic proteins. This was associated with a lower apoptosis rate than those from controls evidenced by delayed phosphatidylserine externalization and mitochondria depolarization. In addition, inflammatory neutrophils from CF patients lungs showed an intense coronin-1 immunolabeling. We concluded that coronin-1 could constitute a potential target in resolving inflammation.

PR3, also called myeloblastin, is a neutrophil serine protease that promotes myeloid cell proliferation by cleaving the cyclin-dependent kinase inhibitor p21(cip1/waf1). In addition, it is the target of ANCA in GPA, a necrotizing vasculitis. Anti-PR3 ANCA binding to membrane-expressed PR3 triggers neutrophil activation, potentiating vascular inflammation. This study performed in RBL cells identifies the structural motifs of PR3 membrane anchorage and examines its impact on PR3 proinflammatory and proliferative functions. With the use of MD simulations and mutagenesis, we demonstrate that the mutations of four hydrophobic (F180, F181, L228, F229) or four basic (R193, R194, K195, R227) amino acids abrogated PR3 membrane anchorage. The hydrophobic patch-deficient PR3 mutant (PR34H4A) was still able to cleave the synthetic substrate Boc-Ala-Pro-Val in cell lysates. However, in contrast to WT PR3, PR34H4A was not expressed at the plasma membrane after degranulation and failed to cleave extracellular fibronectin, was not externalized after apoptosis and did not impair macrophage phagocytosis of apoptotic cells, did not promote myeloid cell proliferation and failed to cleave p21/waf1. PR3 membrane insertion appears to be pivotal for its proinflammatory activities, such as extracellular proteolysis and impairment of apoptotic cell clearance, but also for myeloid cell proliferation. Targeting membrane-associated PR3 might constitute a novel, anti-inflammatory therapeutic strategy in inflammatory disease especially in vasculitis, but this approach has to be validated in mature neutrophils.

In the present study, we provide evidence that procaspase-3 is a novel target of proteinase 3 (PR3) but not of human neutrophil elastase (HNE). Human mast cell clone 1 (HMC1) and rat basophilic leukemia (RBL) mast cell lines were transfected with PR3 or the inactive mutated PR3 (PR3S203A) or HNE cDNA. In both RBL/PR3 and HMC1/PR3, a constitutive activity of caspase-3 was measured with DEVD substrate, due to the direct processing of procaspase-3 by PR3. No caspase-3 activation was observed in cells transfected with the inactive PR3 mutant or HNE. Despite the high caspase-3 activity in RBL/PR3, no apoptosis was detected as demonstrated by an absence of 1) phosphatidylserine externalization, 2) mitochondria cytochrome c release, 3) upstream caspase-8 or caspase-9 activation, or 4) DNA fragmentation. In vitro, purified PR3 cleaved procaspase-3 into an active 22-kDa fragment. In neutrophils, the 22-kDa caspase-3 activation fragment was present only in resting neutrophils but was absent after apoptosis. The 22 kDa fragment was specific of myeloid cells because it was absent from resting lymphocytes. This 22-kDa fragment was not present when neutrophils were treated with pefabloc, an inhibitor of serine proteinase. Like in HMC1/PR3, the 22-kDa caspase-3 fragment was restricted to the plasma membrane compartment. Double immunofluorescence labeling after streptolysin-O permeabilization further showed that PR3 and procaspase-3 could colocalize in an extragranular compartment. In conclusion, our results strongly suggest that compartmentalized PR3-induced caspase-3 activation might play specific functions in neutrophil survival.

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