Myeloperoxidase-specific antineutrophil cytoplasmic antibody-associated vasculitis.

Myeloperoxidase (MPO)-specific antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (MPO-ANCA-associated vasculitis) is one of two major ANCA-associated vasculitis variants characterised by systemic necrotising vasculitis with few or no immune deposits. MPO-ANCA-associated vasculitis predominantly affects small blood vessels and, in contrast to its counterpart proteinase 3-ANCA-associated vasculitis, is generally not associated with granulomatous inflammation. The kidneys and lungs are the most commonly affected organs. The pathogenesis of MPO-ANCA-associated vasculitis is characterised by loss of tolerance to the neutrophil enzyme MPO. This loss of tolerance leads to a chronic immunopathological response where neutrophils become both the target and effector of autoimmunity. MPO-ANCA drives neutrophil activation, leading in turn to tissue and organ damage. Clinical trials have improved the therapeutic approach to MPO-ANCA-associated vasculitis. However, there remains substantial unmet need regarding relapse frequency, toxicity of current treatment, and long-term morbidity. In this Series paper, we present the current state of research regarding pathogenesis, diagnosis, and treatment of MPO-ANCA-associated vasculitis.

G-CSF reshapes the cytosolic PCNA scaffold and modulates glycolysis in neutrophils.

Cytosolic proliferating cell nuclear antigen (PCNA) is involved in neutrophil survival and function, in which it acts as a scaffold and associates with proteins involved in apoptosis, NADPH oxidase activation, cytoskeletal dynamics, and metabolism. While the PCNA interactome has been characterized in neutrophils under homeostatic conditions, less is known about neutrophil PCNA in pathophysiological contexts. Granulocyte colony-stimulating factor (G-CSF) is a cytokine produced in response to inflammatory stimuli that regulates many aspects of neutrophil biology. Here, we used isolated normal-density neutrophils from G-CSF-treated haemopoietic stem cell donors (GDs) as a model to understand the role of PCNA during inflammation. Proteomic analysis of the neutrophil cytosol revealed significant differences between GDs and healthy donors (HDs). PCNA was one of the most upregulated proteins in GDs, and the PCNA interactome was significantly different in GDs compared with HDs. Importantly, while PCNA associated with almost all enzymes involved in glycolysis in HDs, these associations were decreased in GDs. Functionally, neutrophils from GDs had a significant increase in glycolysis compared with HDs. Using p21 competitor peptides, we showed that PCNA negatively regulates neutrophil glycolysis in HDs but had no effect on GD neutrophils. These data demonstrate that G-CSF alters the PCNA scaffold, affecting interactions with key glycolytic enzymes, and thus regulates glycolysis, the main energy pathway utilized by neutrophils. By this selective control of glycolysis, PCNA can organize neutrophils functionality in parallel with other PCNA mechanisms of prolonged survival. PCNA may therefore be instrumental in the reprogramming that neutrophils undergo in inflammatory or tumoral settings.

Dysregulation of neutrophil oxidant production and interleukin-1-related cytokines in granulomatosis with polyangiitis.

OBJECTIVES: Neutrophils play a key role in ANCA-associated vasculitis, both as targets of autoimmunity and facilitators of vascular damage. In granulomatosis with polyangiitis (GPA), data regarding the production of reactive oxygen species (ROS) in neutrophils are unclear. Further, recent data suggests that ROS production could have an anti-inflammatory effect through the regulation of the inflammasome and IL-1-related cytokines. We aimed to analyse the ROS production in neutrophils from patients with GPA and investigate its association with IL-1-related cytokines and the autoantigen proteinase 3 (PR3). METHODS: Seventy-two GPA patients with disease flare were included in the NEUTROVASC prospective cohort study. ROS production was evaluated in whole blood of patients with active GPA and compared with the same patients in remission or healthy controls. Associations between ROS production, PR3 membrane expression on neutrophils, serum levels of IL-1-related cytokines as well as inflammasome-related proteins were analyzed. RESULTS: We observed a robust defect in ROS production by neutrophils from patients with active GPA compared with healthy controls, independent of glucocorticoid treatment. Serum levels of IL-1-related cytokines were significantly increased in GPA patients, particularly in patients with kidney involvement, and levels of these cytokines returned to normal after patients achieved remission. Further, inflammasome-related proteins were significantly dysregulated in the cytosol of neutrophils as well as the serum from GPA patients. CONCLUSION: Our data suggests that ROS production and regulation of the inflammasome in neutrophils from patients with GPA are disturbed and may be a potential therapeutic target. CLINICAL TRIAL REGISTRATION NUMBER: NCT01862068, clinicaltrials.gov, https://www.clinicaltrials.gov.

Thrombopoietin-Dependent Myelo-Megakaryopoiesis Fuels Thromboinflammation and Worsens Antibody-Mediated Chronic Renal Microvascular Injury.

SIGNIFICANCE STATEMENT: Kidney-derived thrombopoietin (TPO) increases myeloid cell and platelet production during antibody-mediated chronic kidney disease (AMCKD) in a mouse model, exacerbating chronic thromobinflammation in microvessels. The effect is mirrored in patients with extracapillary glomerulonephritis associated with thromboinflammation, TGF ß -dependent glomerulosclerosis, and increased bioavailability of TPO. Neutralization of TPO in mice normalized hematopoiesis, reduced chronic thromboinflammation, and ameliorated renal disease. The findings suggest that TPO is a relevant biomarker and a promising therapeutic target for patients with CKD and other chronic thromboinflammatory diseases.Neutralization of TPO in mice normalized hematopoiesis, reduced chronic thromboinflammation, and ameliorated renal disease. The findings suggest that TPO is a relevant biomarker and a promising therapeutic target for patients with CKD and other chronic thromboinflammatory diseases. BACKGROUND: Chronic thromboinflammation provokes microvascular alterations and rarefaction, promoting organ dysfunction in individuals with various life-threatening diseases. Hematopoietic growth factors (HGFs) released by the affected organ may sustain emergency hematopoiesis and fuel the thromboinflammatory process. METHODS: Using a murine model of antibody-mediated chronic kidney disease (AMCKD) and pharmacological interventions, we comprehensively monitored the response to injury in the circulating blood, urine, bone marrow, and kidney. RESULTS: Experimental AMCKD was associated with chronic thromboinflammation and the production of HGFs, especially thrombopoietin (TPO), by the injured kidney, which stimulated and skewed hematopoiesis toward myelo-megakaryopoiesis. AMCKD was characterized by vascular and kidney dysfunction, TGF ß -dependent glomerulosclerosis, and microvascular rarefaction. In humans, extracapillary glomerulonephritis is associated with thromboinflammation, TGF ß -dependent glomerulosclerosis, and increased bioavailability of TPO. Analysis of albumin, HGF, and inflammatory cytokine levels in sera from patients with extracapillary glomerulonephritis allowed us to identify treatment responders. Strikingly, TPO neutralization in the experimental AMCKD model normalized hematopoiesis, reduced chronic thromboinflammation, and ameliorated renal disease. CONCLUSION: TPO-skewed hematopoiesis exacerbates chronic thromboinflammation in microvessels and worsens AMCKD. TPO is both a relevant biomarker and a promising therapeutic target in humans with CKD and other chronic thromboinflammatory diseases.

Specific circulating neutrophils subsets are present in clinically stable adults with cystic fibrosis and are further modulated by pulmonary exacerbations.

The progressive lung destruction in cystic fibrosis (CF) is tightly associated with chronic bacterial infection and neutrophil-dominated airway inflammation. CF pulmonary disease is complicated by episodes of acute exacerbations, contributing to irreversible lung damage. We hypothesized that circulating subsets of neutrophils from clinically stable adults with CF present some phenotypic specificities that could amplify their activation during an infectious episode. The aim of the present study was to examine the different neutrophil subsets in whole blood and in the low density neutrophils (LDN) that co-purify with peripheral blood mononuclear cells (PBMC) in clinically stable adults with CF and in CF adults during pulmonary exacerbations compared to healthy donors. Blood samples were obtained from 22 adults with CF (16 in stable state and 6 during pulmonary exacerbations) and from 20 healthy donors. Flow cytometry analysis of 13 different markers related to lineage (CD45, CD15), maturity (CD16, CD10, and CD33), activation (CD62L, CD11b, CD66b, and CD114), metabolism (GLUT-1, LOX1) and immunosuppression (PD1, PD-L1) was carried out within whole blood and within the LDN fraction. Unsupervised analysis of flow cytometry data was performed using visual t-distributed stochastic neighbor embedding (vi-tSNE). A significant increase in the CD11b expression in neutrophils from CF patients during exacerbations was observed compared to neutrophils from stable CF patients or to healthy donors, indicative of a circulating activation state due to an infectious status. The percentage of LDN was not increased in stable CF patients but increased during exacerbations. Analysis of neutrophil subsets using the double CD16/CD62L labeling revealed a significant increase in the CD16high/CD62Llow subset in all CF patients compared to healthy donors. In contrast, an increase in the CD16low/CD62Lhigh subset was observed only in CF patients during exacerbations. Unsupervised analysis identified a PD-L1high/CD114high population that was present in stable CF patients and as well as in CF patients during exacerbations.

G-CSF - A double edge sword in neutrophil mediated immunity.

Neutrophils are vital for the innate immune system's control of pathogens and neutrophil deficiency can render the host susceptible to life-threatening infections. Neutrophil responses must also be tightly regulated because excessive production, recruitment or activation of neutrophils can cause tissue damage in both acute and chronic inflammatory diseases. Granulocyte colony stimulating factor (G-CSF) is a key regulator of neutrophil biology, from production, differentiation, and release of neutrophil precursors in the bone marrow (BM) to modulating the function of mature neutrophils outside of the BM, particularly at sites of inflammation. G-CSF acts by binding to its cognate cell surface receptor on target cells, causing the activation of intracellular signalling pathways mediating the proliferation, differentiation, function, and survival of cells in the neutrophil lineage. Studies in humans and mice demonstrate that G-CSF contributes to protecting the host against infection, but conversely, it can play a deleterious role in inflammatory diseases. As such, neutrophils and the G-CSF pathway may provide novel therapeutic targets. This review will focus on understanding the role G-CSF plays in the balance between effective neutrophil mediated host defence versus neutrophil-mediated inflammation and tissue damage in various inflammatory and infectious diseases.

Skewed peripheral B- and T-cell compartments in patients with ANCA-associated vasculitis.

OBJECTIVES: To characterize lymphocytes dysregulation in patients with granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). METHODS: Using flow cytometry, we analysed B- and T-cell subsets in peripheral blood from 37 untreated patients with active disease (29 GPA and 8 MPA) and 22 healthy controls (HCs). RESULTS: GPA patients had increased Th2 (1.8 vs 1.0%, P = 0.02), Th9 (1.1 vs 0.2%, P = 0.0007) and Th17 (1.4 vs 0.9%, P = 0.03) cells compared with HC. Patients with MPO-ANCAs had significantly more CD21- B cells than HC or PR3-ANCA patients (6.9 vs 3.3% and 4.4%, P = 0.01). CD69 expressing B cells were significantly higher in GPA and MPA (3.0 and 5.9 vs 1.4%, P = 0.02 and P = 0.03, respectively) compared with HC, whereas B-cell activating factor-receptor expression was decreased in GPA and MPA (median fluorescence intensity ratio 11.8 and 13.7 vs 45.1 in HC, P < 0.0001 and P = 0.003, respectively). Finally, IL-6-producing B cells were increased in GPA vs HC (25.8 vs 14.9%, P < 0.0001) and decreased in MPA vs HC (4.6 vs 14.9%, P = 0.005), whereas TNF-α-producing B cells were lower in both GPA and MPA patients compared with controls (15 and 8.4 vs 30%, P = 0.01 and P = 0.006, respectively). CONCLUSION: Skewed T-cell polarization towards Th2, Th9 and Th17 responses characterizes GPA, whereas B-cell populations are dysregulated in both GPA and MPA with an activated phenotype and a decreased B-cell activating factor-receptor expression. Finally, inflammatory B cells producing IL-6 are dramatically increased in GPA, providing an additional mechanism by which rituximab could be effective.

Cytosolic PCNA interacts with p47phox and controls NADPH oxidase NOX2 activation in neutrophils.

Neutrophils produce high levels of reactive oxygen species (ROS) by NADPH oxidase that are crucial for host defense but can lead to tissue injury when produced in excess. We previously described that proliferating cell nuclear antigen (PCNA), a nuclear scaffolding protein pivotal in DNA synthesis, controls neutrophil survival through its cytosolic association with procaspases. We herein showed that PCNA associated with p47phox, a key subunit of NADPH oxidase, and that this association regulated ROS production. Surface plasmon resonance and crystallography techniques demonstrated that the interdomain-connecting loop of PCNA interacted directly with the phox homology (PX) domain of the p47phox. PCNA inhibition by competing peptides or by T2AA, a small-molecule PCNA inhibitor, decreased NADPH oxidase activation in vitro. Furthermore, T2AA provided a therapeutic benefit in mice during trinitro-benzene-sulfonic acid (TNBS)-induced colitis by decreasing oxidative stress, accelerating mucosal repair, and promoting the resolution of inflammation. Our data suggest that targeting PCNA in inflammatory neutrophils holds promise as a multifaceted antiinflammatory strategy.

Proteomic analysis of neutrophils in ANCA-associated vasculitis reveals a dysregulation in proteinase 3-associated proteins such as annexin-A1 involved in apoptotic cell clearance.

Granulomatosis with polyangiitis (GPA) is an autoimmune vasculitis associated with anti-neutrophil-cytoplasmic antibodies (ANCA) against proteinase 3 leading to kidney damage. Neutrophils from those patients have increased expression of membrane proteinase 3 during apoptosis. Here we examined whether neutrophils from patients with GPA have dysregulated protein expressions associated with apoptosis. A global proteomic analysis was performed comparing neutrophils from patients with GPA, with healthy individuals under basal conditions and during apoptosis. At disease onset, the cytosolic proteome of neutrophils of patients with GPA before treatment was significantly different from healthy controls, and this dysregulation was more pronounced following ex vivo apoptosis. Proteins involved in cell death/survival were altered in neutrophils of patients with GPA. Several proteins identified were PR3-binding partners involved in the clearance of apoptotic cells, namely calreticulin, annexin-A1 and phospholipid scramblase 1. These proteins form a platform at the membrane of apoptotic neutrophils in patients with GPA but not healthy individuals and this was associated with the clinical presentation of GPA. Thus, our study shows that neutrophils from patients with GPA have an intrinsic dysregulation in proteins involved in apoptotic cell clearance, which could contribute to the unabated inflammation and autoimmunity in GPA. Hence, harnessing these dysregulated pathways could lead to novel biomarkers and targeted therapeutic opportunities to treat kidney disease.

Regulation of macrophage activation by proteins expressed on apoptotic neutrophils: Subversion towards autoimmunity by proteinase 3.

Neutrophils are critically involved in host defence and they also modulate the inflammatory process. Turning the inflammatory response towards a resolutive outcome requires a dialogue between apoptotic neutrophils and proresolving macrophages through complex key molecular interactions controlling efferocytosis, anti-inflammatory reprogramming and ultimately immune regulation. In this review, we will first focus on recent molecular analyses aiming at characterizing the role of proteins expressed on apoptotic neutrophils and their cognate partners expressed on macrophages in the resolution of inflammation. These will include chemokine receptors and their ligands and annexin A1 and its receptor FPR2. We will next depict how the structural and enzymatic properties of proteinase 3 (PR3), the autoantigen in vasculitis, allow its expression on apoptotic neutrophils, which in turn affects efferocytosis and immune response associated with the clearance of apoptotic cells. This example illustrates that the fate of apoptotic neutrophils directly influences the resolution of inflammation and immune responses thereby potentially contributing to systemic and nonresolving inflammation as well as autoimmunity.

Proteinase 3 Interferes With C1q-Mediated Clearance of Apoptotic Cells.

Proteinase 3 (PR3) is the autoantigen in granulomatosis with polyangiitis, an autoimmune necrotizing vasculitis associated with anti-neutrophil cytoplasmic antibodies (ANCAs). Moreover, PR3 is a serine protease whose membrane expression can potentiate inflammatory diseases such as ANCA-associated vasculitis and rheumatoid arthritis. During apoptosis, PR3 is co-externalized with phosphatidylserine (PS) and is known to modulate the clearance of apoptotic cells through a calreticulin (CRT)-dependent mechanism. The complement protein C1q is one mediator of efferocytosis, the clearance of altered self-cells, particularly apoptotic cells. Since PR3 and C1q are both involved in the clearance of apoptotic cells and immune response modulation and share certain common ligands (i.e., CRT and PS), we examined their possible interaction. We demonstrated that C1q binding was increased on apoptotic rat basophilic leukemia (RBL) cells that expressed PR3, and we demonstrated the direct interaction between purified C1q and PR3 molecules as shown by surface plasmon resonance. To better understand the functional consequence of this partnership, we tested C1q-dependent phagocytosis of the RBL cell line expressing PR3 and showed that PR3 impaired C1q enhancement of apoptotic cell uptake. These findings shed new light on the respective roles of C1q and PR3 in the elimination of apoptotic cells and suggest a novel potential axis to explore in autoimmune diseases characterized by a defect in apoptotic cell clearance and in the resolution of inflammation.

Granulomatosis with polyangiitis (Wegener granulomatosis): A proteinase-3 driven disease?

Granulomatosis with polyangiitis (GPA, Wegener granulomatosis) is a systemic autoimmune vasculitis that affects small arteries, arterioles, and capillaries, most notably in the kidneys and lungs. In this disease, proteinase-3 (PR3), produced by neutrophils, is targeted by antineutrophil cytoplasmic antibodies (ANCA). Recent work by our group has shown how PR3 impairs the resolution of inflammation and deregulates the immune system. Normally, the clearance of activated neutrophils triggers an anti-inflammatory, pro-resolution process. In patients with GPA, however, macrophages phagocytose apoptotic neutrophils then release massive amounts of pro-inflammatory mediators, notably interleukin-1, thereby generating a pro-inflammatory microenvironment conducive to autoimmunity. This deregulation of immune processes is accompanied with activation of plasmacytoid dendritic cells and with polarization of T-helper-2 (Th2), Th9, and Th17 cells. These recent data highlight the dual role of PR3, both auto-antigenic and auto-inflammatory, thus potentially opening up new therapeutic avenues.

Harnessing Neutrophil Survival Mechanisms during Chronic Infection by Pseudomonas aeruginosa: Novel Therapeutic Targets to Dampen Inflammation in Cystic Fibrosis.

More than two decades after cloning the cystic fibrosis transmembrane regulator (CFTR) gene, the defective gene in cystic fibrosis (CF), we still do not understand how dysfunction of this ion channel causes lung disease and the tremendous neutrophil burden which persists within the airways; nor why chronic colonization by Pseudomonas aeruginosa develops in CF patients who are thought to be immunocompetent. It appears that the microenvironment within the lung of CF patients provides favorable conditions for both P. aeruginosa colonization and neutrophil survival. In this context, the ability of bacteria to induce hypoxia, which in turn affects neutrophil survival is an additional level of complexity that needs to be accounted for when controlling neutrophil fate in CF. Recent studies have underscored the importance of neutrophils in innate immunity and their functions appear to extend far beyond their well-described role in antibacterial defense. Perhaps a disturbance in neutrophil reprogramming during the course of an infection severely modulates the inflammatory response in CF. Furthermore there is an emerging concept that the CFTR itself may be an immune modulator and stimulating CFTR function in CF patients could promote neutrophil and macrophages antimicrobial function. Fostering the resolution of inflammation by favoring neutrophil apoptosis could preserve their microbicidal activities but decrease their proinflammatory potential. In this context, triggering neutrophil apoptosis with roscovitine may be a potential therapeutic option and this is currently being evaluated in CF patients. In the present review we discuss how neutrophils functions are disturbed in CF and how this may relate to chronic infection with P. aeuginosa and we propose novel research directions aimed at modulating neutrophil survival, dampening lung inflammation and ultimately leading to an amelioration of the lung disease.

Proteinase 3: the odd one out that became an autoantigen.

Neutrophils are critical in the defense against bacterial and fungal pathogens, and they also modulate the inflammatory process. The areas where neutrophils are studied have expanded from the restricted field of antibacterial defense to the modulation of inflammation and finally, to fine-tuning immune responses. As a result, recent studies have shown that neutrophils are implicated in several systemic autoimmune diseases, although exactly how neutrophils contribute to these diseases and the molecular mechanisms responsible are still under investigation. In a group of autoimmune vasculitides associated with anti-neutrophil cytoplasmic antibodies (AAVs), granulomatosis with polyangiitis (GPA) illustrates the concept that autoimmunity can develop against one specific neutrophil protein, namely, proteinase 3 (PR3), one of the four serine protease homologs contained within azurophilic granules. In this review, we will focus on recent molecular analyses combined with functional studies that provide clear evidence that the pathogenic properties of PR3 are not only a result of its enzymatic activity but also mediated by a particular structural element-the hydrophobic patch-which facilitates associations with various proteins and lipids and permits anchorage into the plasma membrane. Furthermore, these unique structural and functional characteristics of PR3 might be key contributors to the systemic inflammation and to the immune dysregulation observed in GPA.

Molecular analysis of vascular smooth muscle cells from patients with giant cell arteritis: Targeting endothelin-1 receptor to control proliferation.

OBJECTIVE: The pathophysiology of giant cell arteritis (GCA) and the mechanisms underlying vascular remodeling, are poorly understood. We aimed to compare vascular smooth muscle cells (VSMCs) from patients with GCA and controls by a proteomic and gene expression profile approach and to identify the signaling pathways involved in proliferation. METHODS: VSMCs were cultured from temporal artery biopsies (TABs) from patients with biopsy-proven GCA (TAB+-GCA), biopsy-negative GCA (TAB--GCA), and diagnosis other than GCA (GCA-control). VSMCs from normal human aorta (HAoSMC) were used as controls. 2D-differential in-gel electrophoresis and Affymetrix chips were used to compare proteomes and gene expression profiles of VSMCs. Proliferation was assessed by BrdU incorporation assay. TAB+-GCA and GCA-control TABs underwent immunohistochemistry staining for endothelin-1 (ET-1) and receptors ETAR and ETBR. RESULTS: We identified 16, 30 and 2 protein spots differentially expressed between TAB+-GCA and GCA-control VSMCs, TAB+-GCA and TAB--GCA VSMCs and TAB--GCA and GCA-control VSMCs, respectively (fold change ≥1.5 and p≤0.05). Among the 153 proteins differentially expressed between TAB+-GCA and HAoSMC VSMCs, many were linked with ET-1. Genes differentially expressed between TAB+-GCA and GCA-control VSMCs were involved in proliferation. ET-1 was identified as a link between genes of interest. Proliferation was reduced for TAB+-GCA VSMCs on treatment with the endothelin antagonist macitentan and its active metabolite. Patients showing transmural expression of ET-1 in temporal artery lesions received a significantly higher glucocorticoid daily dose after 6-month follow-up. CONCLUSION: Inhibiting the proliferation with macitentan, combined with glucocorticoids, might be a promising therapeutic approach for patients with GCA.

Cytoplasmic proliferating cell nuclear antigen connects glycolysis and cell survival in acute myeloid leukemia.

Cytosolic proliferating cell nuclear antigen (PCNA), a scaffolding protein involved in DNA replication, has been described as a key element in survival of mature neutrophil granulocytes, which are non-proliferating cells. Herein, we demonstrated an active export of PCNA involved in cell survival and chemotherapy resistance. Notably, daunorubicin-resistant HL-60 cells (HL-60R) have a prominent cytosolic PCNA localization due to increased nuclear export compared to daunorubicin-sensitive HL-60 cells (HL-60S). By interacting with nicotinamide phosphoribosyltransferase (NAMPT), a protein involved in NAD biosynthesis, PCNA coordinates glycolysis and survival, especially in HL-60R cells. These cells showed a dramatic increase in intracellular NAD+ concentration as well as glycolysis including increased expression and activity of hexokinase 1 and increased lactate production. Furthermore, this functional activity of cytoplasmic PCNA was also demonstrated in patients with acute myeloid leukemia (AML). Our data uncover a novel pathway of nuclear export of PCNA that drives cell survival by increasing metabolism flux.

Proteinase 3 Is a Phosphatidylserine-binding Protein That Affects the Production and Function of Microvesicles.

Proteinase 3 (PR3), the autoantigen in granulomatosis with polyangiitis, is expressed at the plasma membrane of resting neutrophils, and this membrane expression increases during both activation and apoptosis. Using surface plasmon resonance and protein-lipid overlay assays, this study demonstrates that PR3 is a phosphatidylserine-binding protein and this interaction is dependent on the hydrophobic patch responsible for membrane anchorage. Molecular simulations suggest that PR3 interacts with phosphatidylserine via a small number of amino acids, which engage in long lasting interactions with the lipid heads. As phosphatidylserine is a major component of microvesicles (MVs), this study also examined the consequences of this interaction on MV production and function. PR3-expressing cells produced significantly fewer MVs during both activation and apoptosis, and this reduction was dependent on the ability of PR3 to associate with the membrane as mutating the hydrophobic patch restored MV production. Functionally, activation-evoked MVs from PR3-expressing cells induced a significantly larger respiratory burst in human neutrophils compared with control MVs. Conversely, MVs generated during apoptosis inhibited the basal respiratory burst in human neutrophils, and those generated from PR3-expressing cells hampered this inhibition. Given that membrane expression of PR3 is increased in patients with granulomatosis with polyangiitis, MVs generated from neutrophils expressing membrane PR3 may potentiate oxidative damage of endothelial cells and promote the systemic inflammation observed in this disease.