CD14 expression is increased on monocytes in patients with anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis and correlates with the expression of ANCA autoantigens.

Monocyte subsets with differing functional properties have been defined by their expression of CD14 and CD16. We investigated these subsets in anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) and determined their surface expression of ANCA autoantigens. Flow cytometry was performed on blood from 14 patients with active AAV, 46 patients with AAV in remission and 21 controls. The proportion of classical (CD14(high) CD16(neg/low)), intermediate (CD14(high) CD16(high)) and non-classical (CD14(low) CD16(high)) monocytes and surface expression levels of CD14 and CD16 were determined, as well as surface expression of proteinase 3 (PR3) and myeloperoxidase (MPO) on monocyte subsets. There was no change in the proportion of monocytes in each subset in patients with AAV compared with healthy controls. The expression of CD14 on monocytes from patients with active AAV was increased, compared with patients in remission and healthy controls (P < 0.01). Patients with PR3-ANCA disease in remission also had increased monocyte expression of CD14 compared with controls (P < 0.01); however, levels in patients with MPO-ANCA disease in remission were lower than active MPO-ANCA patients, and not significantly different from controls. There was a correlation between CD14 and both PR3 and MPO expression on classical monocytes in AAV patients (r = 0.79, P < 0.0001 and r = 0.42, P < 0.005, respectively). In conclusion, there was an increase in monocyte CD14 expression in active AAV and PR3-ANCA disease in remission. The correlation of CD14 expression with ANCA autoantigen expression in AAV may reflect cell activation, and warrants further investigation into the potential for increased CD14 expression to trigger disease induction or relapse.

Proteinase 3 contributes to transendothelial migration of NB1-positive neutrophils.

Neutrophil transmigration requires the localization of neutrophils to endothelial cell junctions, in which receptor-ligand interactions and the action of serine proteases promote leukocyte diapedesis. NB1 (CD177) is a neutrophil-expressed surface molecule that has been reported to bind proteinase 3 (PR3), a serine protease released from activated neutrophils. PR3 has demonstrated proteolytic activity on a number of substrates, including extracellular matrix proteins, although its role in neutrophil transmigration is unknown. Recently, NB1 has been shown to be a heterophilic binding partner for the endothelial cell junctional protein, PECAM-1. Disrupting the interaction between NB1 and PECAM-1 significantly inhibits neutrophil transendothelial cell migration on endothelial cell monolayers. Because NB1 interacts with endothelial cell PECAM-1 at cell junctions where transmigration occurs, we considered that NB1-PR3 interactions may play a role in aiding neutrophil diapedesis. Blocking Abs targeting the heterophilic binding domain of PECAM-1 significantly inhibited transmigration of NB1-positive neutrophils through IL-1ß-stimulated endothelial cell monolayers. PR3 expression and activity were significantly increased on NB1-positive neutrophils following transmigration, whereas neutrophils lacking NB1 demonstrated no increase in PR3. Finally, using selective serine protease inhibitors, we determined that PR3 activity facilitated transmigration of NB1-positive neutrophils under both static and flow conditions. These data demonstrate that PR3 contributes in the selective recruitment of the NB1-positive neutrophil population.

Characterization of chronic myeloid leukemia stem cells.

Although tyrosine kinase inhibitors have redefined the care of chronic myeloid leukemia (CML), these agents have not proved curative, likely due to resistance of the leukemia stem cells (LSC). While a number of potential therapeutic targets have emerged in CML, their expression in the LSC remains largely unknown. We therefore isolated subsets of CD34(+) stem/progenitor cells from normal donors and from patients with chronic phase or blast crisis CML. These cell subsets were then characterized based on ability to engraft immunodeficient mice and expression of candidate therapeutic targets. The CD34(+)CD38(-) CML cell population with high aldehyde dehydrogenase (ALDH) activity was the most enriched for immunodeficient mouse engrafting capacity. The putative targets: PROTEINASE 3, SURVIVIN, and hTERT were expressed only at relatively low levels by the CD34(+)CD38(-)ALDH(high) CML cells, similar to the normal CD34(+)CD38(-)ALDH(high) cells and less than in the total CML CD34(+) cells. In fact, the highest expression of these antigens was in normal, unfractionated CD34(+) cells. In contrast, PRAME and WT1 were more highly expressed by all CML CD34(+) subsets than their normal counterparts. Thus, ALDH activity appears to enrich for CML stem cells, which display an expression profile that is distinct from normal stem/progenitor cells and even the CML progenitors. Indeed, expression of a putative target by the total CD34(+) population in CML does not guarantee expression by the LSC. These expression patterns suggest that PROTEINASE 3, SURVIVIN, and hTERT are not optimal therapeutic targets in CML stem cells; whereas PRAME and WT1 seem promising.

Elevated neutrophil membrane expression of proteinase 3 is dependent upon CD177 expression.

Proteinase 3 (PR3) is a major autoantigen in anti-neutrophil cytoplasmic antibodies (ANCA)-associated systemic vasculitis (AASV), and the proportion of neutrophils expressing PR3 on their membrane (mPR3+) is increased in AASV. We have shown recently that mPR3 and CD177 are expressed on the same cells in healthy individuals. In this study we try to elucidate mechanisms behind the increased mPR3 expression in AASV and its relationship to CD177. All neutrophils in all individuals were either double-positive or double-negative for mPR3 and CD177. The proportion of double-positive neutrophils was increased significantly in AASV and systemic lupus erythematosus patients. The proportion of mPR3+/CD177+ cells was not correlated to general inflammation, renal function, age, sex, drug treatment and levels of circulating PR3. AASV patients had normal levels of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. Pro-PR3 was found to constitute 10% of circulating PR3 but none of the mPR3. We found increased mRNA levels of both PR3 and CD177 in AASV, but they did not correlate with the proportion of double-positive cells. In cells sorted based on membrane expression, CD177-mRNA was several-fold higher in mPR3+ cells. When exogenous PR3 was added to CD177-transfected U937 cells, only CD177+ cells bound PR3 to their membrane. In conclusion, the increased membrane expression of PR3 found in AASV is not linked directly to circulating PR3 or PR3 gene transcription, but is dependent upon CD177 expression and correlated with the transcription of the CD177 gene.

Proteinase 3 and CD177 are expressed on the plasma membrane of the same subset of neutrophils.

Proteinase 3 (PR3) is found in granules of all neutrophils but also on the plasma membrane of a subset of neutrophils (mPR3). CD177, another neutrophil protein, also displays a bimodal surface expression. In this study, we have investigated the coexpression of these two molecules, as well as the effect of cell activation on their surface expression. We can show that CD177 is expressed on the same subset of neutrophils as mPR3. Experiments show that the expression of mPR3 and CD177 on the plasma membrane is increased or decreased in parallel during cell stimulation or spontaneous apoptosis. Furthermore, we observed a rapid internalization and recirculation of mPR3 and plasma membrane CD177, where all mPR3 is replaced within 30 min. Our findings suggest that the PR3 found on the plasma membrane has its origin in the same intracellular storage as CD177, i.e., secondary granules and secretory vesicles and not primary granules. PR3- and CD177-expressing neutrophils constitute a subpopulation of neutrophils with an unknown role in the innate immune system, which may play an important role in diseases such as Wegener's granulomatosis and polycythemia vera.

Interaction between CD177 and platelet endothelial cell adhesion molecule-1 downregulates membrane-bound proteinase-3 (PR3) expression on neutrophils and attenuates neutrophil activation induced by PR3-ANCA.

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.

Priming by tumor necrosis factor-alpha of human neutrophil NADPH-oxidase activity induced by anti-proteinase-3 or anti-myeloperoxidase antibodies.

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.

Immune functions of proteinase 3.

The primary function of neutrophil-derived serine proteases, neutrophil elastase, cathepsin G, and proteinase 3 (PR3) are thought to be the degradation of extracellular proteins at sites of inflammation, but excessive, prolonged, or inappropriate proteolytic activity causes harmful effects in the body. Although there are strong structural similarities among these proteases, PR3 has unique properties in many respects. In particular, PR3 is a major target antigen of autoantibodies, anti-neutrophil cytoplasmic antibodies (ANCA). Recent findings also revealed that PR3 is critically involved in the regulation of immune function. This review discusses the expression of PR3 in non-hemopoietic cells and focuses on the immune functions of PR3 with respect to direct modulation of cell signaling by PR3, PR3-mediated cell activation, and the possible involvement of protease-activated receptors, modulation of the cytokine network, innate immunity and PR3, and recent findings about the generation and function of ANCA.

Production and applications of recombinant proteinase 3, Wegener's autoantigen: problems and perspectives.

Antineutrophil cytoplasmic antibodies (cANCA) against conformational epitopes ofproteinase 3 (PR3) are regarded as an important pathogenic marker in Wegener's granulomatosis (WG). Hence, PR3-based antibody binding assays are widely used for diagnosis and monitoring of the disease. Purification of the native catalytically active serine protease from granulocytes, however, is relatively inefficient, time-consuming and technically demanding. Conformational changes, partial aggregation, denaturation during purification and contaminations with inhibitors or other proteins from plasma and granulocytes, can affect the quality and comparability of PR3-based cANCA determinations. Alternative production of the human PR3 autoantigen by recombinant technologies offers several advantages over the natural antigen, but the complexity and operating expense of these procedures have, so far, delayed the development of new clinical tests. Correct posttranslational processing, conformational identity and antigen stability can be achieved by the expression of PR3 in Sf9 insect cells and in mammalian hosts, HEK293 and HMC-1. Subsequent purification and immobilization of the recombinant antigen is furthermore simplified by the attachment of short carboxy-terminal peptide tags. In contrast to conventional capture techniques with murine monoclonal antibodies, tag-based immobilization of the recombinant antigen does not mask portions of the PR3 surface and improves the efficacy of antigen coating. Moreover, recombinant PR3 variants will have a great potential to study individual anti-PR3 responses and will advance the development of new epitope-based therapeutics.

Different forms of alpha-1 antitrypsin and neutrophil activation mediated by human anti-PR3 IgG antibodies.

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.

Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis.

Granulomatosis with polyangiitis (GPA) is a systemic necrotizing vasculitis that is associated with granulomatous inflammation and the presence of anti-neutrophil cytoplasmic antibodies (ANCAs) directed against proteinase 3 (PR3). We previously determined that PR3 on the surface of apoptotic neutrophils interferes with induction of antiinflammatory mechanisms following phagocytosis of these cells by macrophages. Here, we demonstrate that enzymatically active membrane-associated PR3 on apoptotic cells triggered secretion of inflammatory cytokines, including granulocyte CSF (G-CSF) and chemokines. This response required the IL-1R1/MyD88 signaling pathway and was dependent on the synthesis of NO, as macrophages from animals lacking these pathways did not exhibit a PR3-associated proinflammatory response. The PR3-induced microenvironment facilitated recruitment of inflammatory cells, such as macrophages, plasmacytoid DCs (pDCs), and neutrophils, which were observed in close proximity within granulomatous lesions in the lungs of GPA patients. In different murine models of apoptotic cell injection, the PR3-induced microenvironment instructed pDC-driven Th9/Th2 cell generation. Concomitant injection of anti-PR3 ANCAs with PR3-expressing apoptotic cells induced a Th17 response, revealing a GPA-specific mechanism of immune polarization. Accordingly, circulating CD4+ T cells from GPA patients had a skewed distribution of Th9/Th2/Th17. These results reveal that PR3 disrupts immune silencing associated with clearance of apoptotic neutrophils and provide insight into how PR3 and PR3-targeting ANCAs promote GPA pathophysiology.

Major histocompatibility complex HLA region largely explains the genetic variance exercised on neutrophil membrane proteinase 3 expression.

ANCA-associated vasculitides, a common cause of rapidly progressive glomerulonephritis, are influenced by genetic variance. Neutrophil membrane expression of the ANCA antigen proteinase 3 (PR3) is pathogenically important. A subset of membrane PR3-positive neutrophils can be distinguished from a membrane-negative subset in any given subject. The percentage of membrane PR3-positive neutrophils is genetically determined. In this study, 17 pairs of HLA-matched siblings were typed for their percentage of membrane PR3-positive neutrophils. The HLA-matched siblings showed a high concordance (r = 0.67, P < 0.05), similar to that seen in monozygotic twins. For testing of whether the HLA system influences membrane PR3 percentage, membrane PR3 typing and HLA typing of 51 unrelated patients with Wegener's granulomatosis and 49 normal control subjects was performed. Using two independent statistical methods, a group of 34 HLA antigens was found to predict a large fraction of the membrane PR3 phenotype in patients and control subjects. Certain major histocompatibility HLA antigens have been implicated to conflicting degrees in ANCA-associated vasculitides. However, in earlier studies, the contribution of the HLA system to the genetic variance of the disease was unclear. In this cohort, found was an association of Wegener's granulomatosis with the same group of HLA antigens that predicted for membrane PR3 percentage and a similar correlation with clinical parameters at initial presentation. The disease status in 80% of the patients and 82% of the control subjects could be predicted correctly on the basis of HLA typing by discriminate function analysis (P < 0.001). After removal of the predicted individual from the sample, this association remained significant (64 and 63% correct prediction; P < 0.001). The data suggest that a complex interaction of the entire HLA system is responsible for the genetic influence on membrane PR3 percentage and Wegener's granulomatosis.