Anti-neutrophil cytoplasmic antibodies (ANCA) from patients with systemic vasculitis recognize restricted epitopes of proteinase 3 involving the catalytic site.
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ANCA with specificity for proteinase 3 (PR3), a neutrophil primary granule enzyme, are of diagnostic value in Wegener's granulomatosis (WG) and certain other forms of systemic vasculitis. There is evidence to suggest that they play a pathogenic role in disease, and that the interaction of ANCA with PR3 is likely to be important. We showed, using a resonant mirror biosensor, that C-ANCA from different patients recognized the same or closely related epitopes on PR3. Studies using linear peptides in the SPOT system confirmed the highly restricted nature of this interaction and identified five linear epitopes. Fluid-phase inhibition studies, using a different set of peptides, validated the sequences involved. Using a computer-generated model of the structure of PR3, four of five epitopes were shown to be intimately linked with the catalytic site. The restricted number of epitopes, and their location at the catalytic site, has important implications for the role of C-ANCA in the pathogenesis of vasculitis. (+info)
Subthreshold concentrations of anti-proteinase 3 antibodies (c-ANCA) specifically prime human neutrophils for fMLP-induced leukotriene synthesis and chemotaxis.
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Anti-neutrophil cytoplasmic antibodies (ANCA) targeting proteinase 3 (PR3) possess a high sensitivity and specificity for Wegener's granulomatosis. Due to their capacity of directly activating neutrophils, a pathogenetic role for these autoantibodies has been proposed. We investigated the impact of subthreshold concentrations of monoclonal anti-PR3 antibodies (anti-PR3; 0.1 microg/mL) on neutrophil activation elicited by a secondary agent. Preincubation with anti-PR3 resulted in a massive amplification of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced leukotriene (LT) generation, with a marked increase in the liberation of LTB4, LTA4, and 5-hydroxyeicosatetraenoic acid (5-HETE). This priming commenced within 2.5 min, with a maximum after 5-7.5 min. Moreover, anti-PR3 pretreatment markedly enhanced PMN movement toward fMLP. The priming effect of anti-PR3 toward fMLP challenge was reproduced by c-ANCA, but not by F(ab)2 fragments of the antibodies and isotype-matched control IgG. Generation of superoxide anion and release of elastase were suppressed in anti-PR3-pretreated neutrophils undergoing fMLP challenge. In contrast, neutrophil activation by platelet-activating factor (PAF) or the calcium ionophore A23187 remained unaffected. We conclude that subthreshold concentrations of anti-PR3 antibodies selectively modify neutrophil responses to fMLP, with enhancement of leukotriene generation and chemotaxis, but suppression of respiratory burst and degranulation. Such priming might contribute to localized neutrophil accumulation together with blunted host defense in Wegener's granulomatosis. (+info)
Analysis of anti-neutrophil cytoplasmic antibodies (ANCA): frequency and specificity in a sample of 191 homozygous (PiZZ) alpha1-antitrypsin-deficient subjects.
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BACKGROUND: ANCA are autoantibodies directed against polymorphonuclear cell antigens, mainly proteinase 3 (PR3) and myeloperoxidase (MPO), which are implicated in the pathogenesis of small-vessel necrotizing vasculitis. Alpha1-antitrypsin is the main inhibitor of neutral serine proteinase [i.e. human leukocyte elastase (HLE) and PR3] present in PMN alpha-granules (alphaGr). An association first reported by us between PR3 ANCA and the deficient PiZZ phenotype in ANCA-positive systemic vasculitis, now widely confirmed by others, led us to study the incidence and specificity of ANCA among PiZZ subjects. METHODS: We tested a population of 191 PiZZ (273 sera) for ANCA activity versus 272 PiMM matched control subjects using alphaGr or antigen-specific ELISA [PR3, HLE, MPO, lactoferin (LF) and bactericidal/ permeability increasing protein (BPI)]. RESULTS: The incidence of antibodies directed against alphaGr and HLE but not PR3, MPO, LF or BPI was increased in the PiZZ as compared to the PiMM group (Fisher probability respectively P < 0.0001 and P < 0.05). CONCLUSIONS: ANCA not directed against classical antigens (MPO and PR3) may be found in PiZZ patients. However, these patients do not develop systemic vasculitis features. Therefore, alpha1-antitrypsin deficiency is not sufficient to induce ANCA positive vasculitides, and may only act as a second hit amplifying factor. (+info)
Antineutrophil cytoplasmic antibodies reacting with the pro form of proteinase 3 and disease activity in patients with Wegener's granulomatosis and microscopic polyangiitis.
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OBJECTIVE: Antineutrophil cytoplasmic antibodies (ANCA) directed against proteinase 3 (PR3) are diagnostic markers for the small vessel vasculitides Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA). Correlation of disease activity with PR3 ANCA levels, as determined by standard methods, is not apparent in every patient. PR3 ANCA react with yet to be identified conformational epitopes. We have identified PR3 ANCA subsets that react differentially with mature recombinant PR3 (rPR3; lacking the N-terminal activation dipeptide) and the pro form of this enzyme (pro-rPR3). The present study was performed to determine the association of these PR3 ANCA subsets with disease activity. METHODS: Sera from 61 PR3 ANCA-positive patients with WG or MPA were assayed by capture enzyme-linked immunosorbent assay using pro-rPR3 and rPR3 as target antigens, and were correlated with disease activity as determined by the Birmingham Vasculitis Activity Score (BVAS). RESULTS: Median levels of PR3 ANCA reacting with pro-rPR3 were higher during active (n = 32) than during inactive (n = 29) disease (P = 0.016). Reactivity with mature rPR3 was not significantly different (P = 0.71). Serial followup in individual patients also indicated better correlation of PR3 ANCA reactivity with pro-rPR3 than with mature rPR3. CONCLUSION: PR3 ANCA subsets reactive with epitopes accessible on pro-PR3 correlate better with disease activity than do subsets reactive with epitopes accessible only on mature PR3. This observation may explain why ANCA levels determined with current standard methods are suboptimal for monitoring disease activity. It raises new questions about the primary target of the PR3 ANCA immune response in patients with small vessel vasculitis. (+info)
Prevalence and spectrum of rheumatic diseases associated with proteinase 3-antineutrophil cytoplasmic antibodies (ANCA) and myeloperoxidase-ANCA.
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OBJECTIVES: To evaluate the prevalence and association of antineutrophil cytoplasmic antibodies (ANCA) and their subtypes [proteinase 3 (PR3)-ANCA, myeloperoxidase (MPO)-ANCA] with distinct clinical features in various clinicopathological syndromes. METHODS: All consecutive ANCA-positive patients seen at the combined unit for rheumatology for Bad Bramstedt and the University of Lubeck between 1989 and 1999 were analysed. ANCA were detected by an immunofluorescence technique and ANCA subspecificities were determined by ELISA. Clinical features at presentation and diagnoses were recorded according to standardized procedures. RESULTS: Among 4620 patients tested, 333 were cytoplasmic ANCA-positive and 291 were perinuclear ANCA-positive. cANCA/PR3-ANCA were strongly associated with Wegener's granulomatosis (WG), whereas pANCA/MPO-ANCA were associated with a diverse disease spectrum. Further investigation of PR3-ANCA-positive (n=80) and MPO-ANCA-positive patients (n=40) revealed a greater extent of disease [disease extent index (DEI); median 8 vs 5, P<0.01] and more frequent involvement of the upper/lower respiratory tract and the eyes in PR3-ANCA-positive than in MPO-ANCA-positive patients. Fewer than 5% of WG patients were MPO-ANCA-positive. Compared with matched PR3-ANCA-positive WG patients, the MPO-ANCA-positive WG patients had a lower DEI (median 5 vs 8) and had a lower frequency of peripheral neuropathy. CONCLUSIONS: ANCA testing is useful due to its high sensitivity and specificity, especially for cANCA/PR3-ANCA in WG. We found a divergence in the disease spectrum between PR3- and MPO-ANCA-positive patients, characterized by higher DEI and extrarenal manifestations in the PR3-ANCA group. MPO-ANCA was rarely found in WG and was associated with less organ involvement. (+info)
Proteinase 3, Wegener's autoantigen: from gene to antigen.
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Proteinase 3 (PR3) is one of four serine protease homologues in the azurophilic granules of neutrophils and granules of monocytes. It is of importance that anti-neutrophil cytoplasmic antibodies (ANCA) in patients with Wegener's granulomatosis (WG) are mainly directed against PR3 only. Furthermore, PR3 is overexpressed in a variety of acute and chronic myeloid leukemia cells. Cytotoxic T lymphocytes specific for a PR3-derived peptide have been shown to specifically lyse leukemia cells that overexpress PR3. This review will focus on PR3 and the characteristics of PR3 that might implicate this particular antigen in the pathogenesis of WG and as target for immunotherapy in myeloid leukemias. We will discuss the genetic localization and gene regulation of PR3, the processing, storage, and expression of the PR3 protein, and the physiological functions of PR3, and compare this with the three other neutrophil-derived serine proteases: human leukocyte elastase, cathepsin G, and azurocidin. Three main differences are described between PR3 and the other serine proteases. This makes PR3 a very intriguing protein with a large array of physiological functions, some of which may play a role in ANCA-associated vasculitidis and myeloid leukemia. (+info)
Myeloblastin is an Myb target gene: mechanisms of regulation in myeloid leukemia cells growth-arrested by retinoic acid.
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A pivotal role has been assigned to Myb in the control of myeloid cell growth. Although Myb is a target of retinoic acid, little is known about the mechanisms by which it may contribute to induced growth arrest in leukemia cells. Indeed, few Myb target genes are known to be linked to proliferation. Myeloblastin is involved in the control of proliferation in myeloid leukemia cells. It is expressed early during hematopoiesis and is a granulocyte colony-stimulating factor-responsive gene. Myeloblastin can confer factor-independent growth to hematopoietic cells, an early step in leukemia transformation. The myeloblastin promoter contains PU.1, C/EBP, and Myb binding sites, each of which are critical for constitutive expression in myeloid cells. Inhibition of myeloblastin expression in leukemia cells growth-arrested by retinoic acid is demonstrated to depend on Myb down-regulation. Myb is shown to induce myeloblastin expression and abolish its down-regulation by retinoic acid. Altogether, the data offer a clue as to how a myeloid-specific transcriptional machinery can be accessible to regulation by retinoic acid and point to myeloblastin as a novel target of Myb. This link between Myb and myeloblastin suggests a previously nonidentified Myb pathway through which growth arrest is induced by retinoic acid in myeloid leukemia cells. (+info)
IgG from myeloperoxidase-antineutrophil cytoplasmic antibody-positive patients stimulates greater activation of primed neutrophils than IgG from proteinase 3-antineutrophil cytosplasmic antibody-positive patients.
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OBJECTIVE: Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA) have been reported to be pathologically and clinically different. The aim of this study was to assess whether these differences could be explained by differing abilities of proteinase 3-antineutrophil cytoplasmic antibody (PR3-ANCA)-positive IgG preparations or myeloperoxidase-ANCA (MPO-ANCA)-positive IgG preparations to activate neutrophils (polymorphonuclear cells [PMN]) in vitro. METHODS: Using Percoll density gradients, PMN were isolated (concentration 2 x 10(6)/ml) and primed with cytochalasin B (1 ng/ml) and tumor necrosis factor alpha (TNFalpha; 2 ng/ml). The PMN were activated with 200 microg/ml of normal IgG or ANCA. Activation was determined by 1) superoxide anion generation as determined by the superoxide dismutase-inhibitable reduction of ferricytochrome c, 2) monitoring fluxes in Ca2+ concentration using Fura 2-AM-loaded PMN, and 3) degranulation using an MPO assay. Surface expression of PR3 and MPO was determined by fluorescence-activated cell sorter analysis. ANCA isotypes were investigated by enzyme-linked immunosorbent assay. RESULTS: Activation of PMN by MPO-ANCA-positive IgG preparations compared with PR3-ANCA-positive IgG preparations resulted in greater generation of superoxide anions (MPO-ANCA-positive IgG preparations 9.13 +/- 0.39 nmoles [mean +/- SEM], PR3-ANCA-positive IgG preparations 6.32 +/- 0.35 nmoles; P < 0.001), Ca2+ fluxes (MPO-ANCA-positive IgG preparations 0.735 +/- 0.10, PR3-ANCA-positive IgG preparations 0.33 +/- 0.098; P < 0.01), and MPO degranulation (MPO-ANCA-positive IgG preparations 251.98 +/- 26.7 ng, PR3-ANCA-positive IgG preparations 145.19 +/- 19.4 ng; P < 0.001). The increased activation seen with MPO-ANCA-positive IgG preparations was not due to increased expression of MPO on the cell surface, because following TNFalpha priming PR3 was expressed on significantly more cells than was MPO (PR3 expression 54.2 +/- 5.18%, MPO 31.6 +/- 3.55%; P < 0.001). IgG1 and IgG4 were the predominant isotypes in both MPO-ANCA-positive IgG preparations and PR3-ANCA. MPO-ANCA contained significantly more IgG1 than did PR3-ANCA, and PR3-ANCA-positive IgG preparations contained significantly more IgG3. CONCLUSION: In vitro MPO-ANCA-positive IgG preparations are more activating than PR3-ANCA-positive IgG preparations. The increased activation cannot be explained by increased MPO expression on the cell surface or greater IgG3 present in MPO-ANCA-positive IgG preparations. Differences in activation of PMN by these antibodies may determine some differences between WG and MPA. (+info)