cathepsin-g and Granulomatosis-with-Polyangiitis

Wegener's granulomatosis belongs to the group of small vessel vasculitis associated with anti-neutrophil cytoplasmic antibodies characterized by granulomatous inflammation and necrotising vasculitis in various organs with particular involvement of the upper and lower respiratory tracts and kidneys. Wegener's granulomatosis is a rare disorder in childhood and early diagnosis of this disease is critical to the long-term prognosis of the disease. The presence of positive cytoplasmic antineutrophil cytoplasmic antibody staining or a high titre of proteinase 3 antibodies were added as new criteria of vasculitis in childhood. This article presents a case of Wegener's granulomatosis, with the presence of anti-neutrophil cytoplasm antibodies with cytoplasmic pattern with absence of anti-proteinase 3 antibodies and presence of high levels of anti-cathepsin G antibodies, rarely described in Wegener's granulomatosis.

Topics: Antibodies, Antineutrophil Cytoplasmic; Cathepsin G; Child; Granulomatosis with Polyangiitis; Humans; Male

Proteinase 3 (PR3), the target antigen of antineutrophil cytoplasm autoantibodies, which are found in patients with Wegener granulomatosis, is a neutrophil serine protease localized within cytoplasmic granules. Recently, the human neutrophil antigen NB1 was identified as a specific neutrophil cell surface receptor of PR3. We hypothesized that the unique hydrophobic cluster of PR3 that is not present on human neutrophil elastase and cathepsin G and presumably is also missing in other human PR3 homologs accounts for its binding to the NB1 receptor expressed on the cellular surface of NB1 cells. Instead of generating and testing various artificial human PR3 mutants, we cloned and expressed the very closely related gibbon (Hylobates pileatus) PR3 homolog, which did not bind to the human NB1 receptor. Moreover, a human-gibbon hybrid constructed from the N- and C-terminal half of the human and gibbon PR3, respectively, also did not interact with human NB1. The C-terminal half of gibbon PR3 differs only by 9 residues from human PR3, among which four closely spaced hydrophobic residues are substituted in a nonconservative manner (F166L, W218R, G219A, and L223H). The NB1-bound PR3 was active and was cleared from the surface by alpha-1-protease inhibitor. Conformational distortion of the hydrophobic 217-225 loop by alpha-1-protease inhibitor most likely triggers rapid solubilization.

Topics: Animals; Antibodies, Antineutrophil Cytoplasmic; Cathepsin G; Cathepsins; Cell Line; GPI-Linked Proteins; Granulomatosis with Polyangiitis; Humans; Hydrophobic and Hydrophilic Interactions; Hylobates; Isoantigens; Leukocyte Elastase; Membrane Glycoproteins; Myeloblastin; Protein Binding; Protein Structure, Secondary; Receptors, Cell Surface; Serine Endopeptidases; Species Specificity

In patients with anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis, indirect immunofluorescence (IF) distinguishes between cytoplasmic (C-ANCA) and perinuclear (P-ANCA) neutrophil staining patterns. In patients with primary systemic vasculitis such as Wegener's granulomatosis, microscopic polyangiitis and Churg-Strauss syndrome, these IF staining patterns correspond broadly with antibodies to the two major antigens: the C-ANCA pattern is associated generally with antibodies to serine protease 3 (PR3) and the P-ANCA pattern with antibodies to myeloperoxidase (MPO). However, some sera positive for ANCA by IF are negative for anti-PR3 and anti-MPO antibodies, suggesting the presence of antibodies to minor antigens of PMN granules. We tested sera from a previously well-defined clinical cohort of patients for antibodies to four possible minor antigens: bactericidal permeability increasing protein, elastase, cathepsin G and lactoferrin. IF-positive (+) sera had significantly higher antibody frequencies to the minor antigens than did the IF-negative (-) sera (P < 0.01). Patients with IF(+) PR3(-)MPO(-) sera showed the most varied reactivity to the minor antigens. Among the IF(+) groups, the IF(+) PR3(+)/MPO(-) sera showed the lowest reactivity to the minor antigens. Patients with well-defined ANCA specificities, e.g. the PR3-ANCA response associated with Wegener's granulomatosis, are less likely than are other patient subsets to have antibodies to minor antigen targets. Autoantibodies to these minor antigens contribute to the overall pattern of ANCA identified by IF and help to explain why the correlation between IF and enzyme immunoassays show discrepancies. While the pathophysiological significance of antibodies to minor target antigens needs further evaluation, they may be markers of inflammation associated with disease processes.

Topics: Antibodies, Antineutrophil Cytoplasmic; Antimicrobial Cationic Peptides; Autoantibodies; Autoantigens; Blood Proteins; Cathepsin G; Cathepsins; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique, Indirect; Granulomatosis with Polyangiitis; Humans; Membrane Proteins; Neoplasm Proteins; Peroxidase; Serine Endopeptidases; Vasculitis

The detection rate of antineutrophil cytoplasmic antibodies (ANCA) in Chinese patients with clinically suspected small vessel vasculitis was investigated, and their antigen specificity and demographic features were analyzed. A number of sera (n = 5,604) sent to our referral laboratory for ANCA screening were tested by indirect immunofluorescence (IIF), enzyme-linked immunosorbent assays (ELISAs) for myeloperoxidase (MPO)- and proteinase 3 (PR3)-ANCA. Then the IIF-ANCA-positive sera that were negative for MPO- and PR3-ANCA were further tested by antigen-specific ELISA by using other five highly purified known ANCA antigens as solid-phase ligands. The known antigens included bactericidal/permeability-increasing protein (BPI), human leukocyte elastase (HLE), lactoferrin, cathepsin G, and azurocidins. Of the 5,604 sera, 267 (4.76%) sera were IIF-ANCA positive and 390 (7%) were antinuclear antibody (ANA) positive in the IIF assay. Of the IIF-positive samples, 213 were anti-MPO positive, 32 were anti-PR3 positive, and five cases were positive for both. Of the 48 sera positive for IIF-ANCA but negative for MPO- and PR3-ANCA, 13 sera (27%) recognized other target antigens, 7 sera recognized BPI, 5 recognized HLE, 1 recognize cathepsin G, and 1 recognized azurocidin. None of the sera recognized lactoferrin, and one serum sample recognized both BPI and HLE. The majority of ANCA-positive patients presented in summer or winter. There was no difference in gender (male/female ratio, 1:1.12) in ANCA-positive patients with a mean age of 53.1 years. The male/female ratio was 1.17:1 for patients over 60 years of age; however, it was 1:4 for patients under 20 years of age. We conclude that ANCA-related diseases are not rare in China, and the major antigens are MPO and PR3. When the IIF technique is used to detect ANCA, ANA should be carefully distinguished.

Topics: Adolescent; Adult; Aged; Anemia, Hemolytic; Antibodies, Antineutrophil Cytoplasmic; Antibodies, Antinuclear; Antibody Specificity; Antimicrobial Cationic Peptides; Autoantibodies; Blood Proteins; Cathepsin G; Cathepsins; Child; China; Colitis, Ulcerative; Enzyme-Linked Immunosorbent Assay; Female; Fluorescent Antibody Technique, Indirect; Granulomatosis with Polyangiitis; Humans; Lactoferrin; Leukocyte Elastase; Male; Membrane Proteins; Middle Aged; Myeloblastin; Peroxidase; Seasons; Serine Endopeptidases; Sex Factors; Vasculitis

The prevalence of antineutrophil cytoplasmic antibodies (ANCA) was studied in 12 children with Wegener's granulomatosis. The serum samples were taken in the active phase of disease and were screened for ANCA by indirect immunofluorescence with normal neutrophils and enzyme linked immunosorbent assay (ELISA) using crude neutrophil extract, proteinase 3, myeloperoxidase, cathepsin G, lactoferrin, and elastase as antigens. Of these 12 patients, 10 wre positive for ANCA in the active phase of their illness, and they showed a predominantly cytoplasmic ANCA staining pattern on indirect immunofluorescence. There were high titres of ANCA directed against crude neutrophil extract, proteinase 3, myeloperoxidase, and cathepsin G. IgM isotypes occurred as commonly as IgG isotypes. Therefore, screening for ANCA is usually but not invariably positive in children with Wegener's granulomatosis. Specific diagnosis still relies on clinical and pathological features, and the value of ANCA in the diagnosis of paediatric Wegener's granulomatosis requires further study.

Topics: Adolescent; Antibodies, Antineutrophil Cytoplasmic; Biomarkers; Cathepsin G; Cathepsins; Child; Child, Preschool; Endopeptidases; Enzyme-Linked Immunosorbent Assay; Epitopes; Female; Fluorescent Antibody Technique, Indirect; Granulomatosis with Polyangiitis; Humans; Immunoglobulin G; Immunoglobulin M; Infant; Male; Peroxidase; Serine Endopeptidases

Proteinase 3 is the major target antigen of antineutrophil cytoplasmic autoantibodies (ANCA) in Wegener's granulomatosis and is contained in the azurophilic granules of polymorphonuclear neutrophils, the dominant cell type in vascular lesions during the early stages of systemic vasculitis. This study questioned whether neutrophil lysosomal enzymes, once released at the site of inflammation, are able to potentiate the influx of additional neutrophils by enhancing the production of the chemotactic cytokine interleukin-8 (IL-8) by endothelial cells. Therefore, human umbilical vein endothelial cells in culture were incubated with varying concentrations of highly purified proteinase 3, human neutrophil elastase, and cathepsin G for different time periods. The supernatants were subsequently assessed for IL-8 antigen by using a sandwich ELISA. The presence of both proteinase 3 and elastase resulted in an increased production of IL-8, up to 15.6- and 4.2-fold, respectively, in a dose- and time-dependent fashion. Cathepsin G did not influence IL-8 production. Although the addition of an alpha 1-proteinase inhibitor completely abrogated elastase-mediated IL-8 production, it did not significantly influence the effect of proteinase 3. Both proteinase 3-and elastase-mediated production of IL-8 was inhibited by cycloheximide, indicating de novo synthesis. This was supported by the finding of increased IL-8 mRNA levels in proteinase 3-treated human umbilical vein endothelial cells by using Northern blot analysis. Taken together, the neutrophil lysosomal enzymes proteinase 3 and human neutrophil elastase may contribute to a self-perpetuating process of neutrophil recruitment in acute inflammation by increasing de novo synthesis of IL-8 by endothelial cells. The studies presented here also show that proteinase 3 mediates its effect independently of its enzymatic activity, indicating a hitherto unknown mode of action on endothelial cells.

Topics: alpha 1-Antitrypsin; Autoantigens; Autoimmune Diseases; Cathepsin G; Cathepsins; Cells, Cultured; Chemotaxis, Leukocyte; Cycloheximide; Endothelium, Vascular; Gene Expression Regulation; Granulomatosis with Polyangiitis; Humans; Interleukin-8; Lysosomes; Myeloblastin; Neutrophils; Protein Synthesis Inhibitors; RNA, Messenger; Serine Endopeptidases; Stimulation, Chemical; Umbilical Veins

Human proteinase-3 is one of three serine proteinases present in the azurophil granules of polymorphonuclear leukocytes along with elastase and cathepsin G. Proteinase-3 gene expression is confined to the promyelocytic stage of polymorphonuclear leukocyte maturation. The present investigation identifies elements responsible for this highly controlled tissue- and developmental-specific expression of proteinase-3. Within the first 200 base pairs of the proteinase-3 promoter, two elements were identified as important for expression, these elements at -101 and -190 confer the majority of the activity. The element at -101 has a PU.1 consensus. It binds a myeloid nuclear protein of approximately 45 kDa that "supershifts" with PU.1 antibody and is competed by the CD11b PU.1 element. The element at -190 has a core sequence of CCCCGCCC (CG element). The cytidines but not the guanidine are essential for promoter activity. The CG element binds a second nuclear protein with a molecular mass of approximately 40 kDa that is found in cells of myeloid lineage as well as non-myeloid HeLa cells. However, the proteinase-3 promoter is not active in HeLa cells which suggests that the CG element alone is not sufficient for proteinase-3 gene expression. Maturation of promyelocytic cells results in an inhibition of proteinase-3 gene expression and a reduction in nuclear protein binding to the PU.1 and CG elements. Similar elements occur in the elastase and cathepsin G promoters. Using the elastase and cathepsin G PU.1 and CG-like elements as probes results in identical band-shift patterns to that obtained with proteinase-3 PU.1 and CG elements. These data suggest that there is cooperative interaction between a PU.1 and a CG element with a consensus of CCCCXCCC and that they are important control elements for tissue- and developmental-specific expression of azurophil serine proteinases of polymorphonuclear leukocytes.

Topics: Autoantigens; Base Sequence; Cathepsin G; Cathepsins; Cell Differentiation; Cytidine; Gene Expression Regulation, Enzymologic; Granulomatosis with Polyangiitis; Humans; Leukocyte Elastase; Molecular Sequence Data; Myeloblastin; Promoter Regions, Genetic; Proto-Oncogene Proteins; Sequence Deletion; Serine Endopeptidases; Tetradecanoylphorbol Acetate; Trans-Activators

Topics: Antibodies, Antineutrophil Cytoplasmic; Antibody Specificity; Autoantibodies; Cathepsin G; Cathepsins; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Epitopes; Granulomatosis with Polyangiitis; Humans; Myeloblastin; Pancreatic Elastase; Sequence Homology, Amino Acid; Serine Endopeptidases

Anti-neutrophil cytoplasmic autoantibodies (ANCA) associated with active Wegener's granulomatosis are directed against a soluble 29-Kd protein present in human neutrophils and monocytes. Affinity labeling with tritiated diisopropylfluorophosphate (3H-DFP) suggested that ANCA-antigen is a serine protease. We used immunoelectron microscopy to study the in situ localization of the ANCA-antigen in normal human neutrophils and monocytes using immunoglobulin G (IgG) from ANCA-positive patients and a mouse monoclonal antibody against the ANCA-antigen. Label was observed on the large granules of the neutrophils and in granules of monocytes. Double-labeling, using anti-myeloperoxidase or the peroxidase reaction as markers for azurophil granules and anti-lactoferrin as marker for specific granules, showed that ANCA is colocalized with markers of azurophil granules but not with lactoferrin. Furthermore, elastase and cathepsin G were found in the azurophil granules of neutrophils and in the peroxidase-positive granules of monocytes, colocalized with ANCA-antigen. Cytochalasin-B-treated neutrophils stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) formed large intracellular vacuoles and were partially degranulated. Some vacuoles contained ANCA-antigen, as well as myeloperoxidase, elastase, and cathepsin G, demonstrating release of these enzymes from the azurophil granules into vacuoles. Our results demonstrate that ANCA-antigen is located in myeloperoxidase-containing granules of neutrophils and monocytes, and is packaged in the same granules as elastase and cathepsin G, the two previously identified serine proteases of myeloid leukocytes.

Topics: Autoantibodies; Autoantigens; Cathepsin G; Cathepsins; Cell Compartmentation; Cytochalasin B; Granulomatosis with Polyangiitis; Humans; Immunoenzyme Techniques; Immunohistochemistry; Lysosomes; Microscopy, Electron; Monocytes; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Pancreatic Elastase; Peroxidase; Serine Endopeptidases; Subcellular Fractions

Anti-neutrophil cytoplasmic autoantibodies (ANCA) specifically associated with Wegener's granulomatosis were found to be directed against a saline-soluble glycoprotein triplet that migrates on SDS gels as distinct bands of Mr 29,000, 30,500, and 32,000 and is present in the azurophilic granules. This antigen was specifically recognized by all cytoplasmic-staining (C)-ANCA-positive sera from patients with Wegener's disease. C-ANCA antigen bound [3H]diisopropylfluorophosphate, which indicates that it is a serine protease, but it could clearly be distinguished from the serine proteases elastase and cathepsin G. Stimulation of cytochalasin B-treated neutrophils with FMLP induced release of C-ANCA antigen. This indicates that in vivo C-ANCA might interact with the C-ANCA antigen after its release upon inflammatory stimulation. We further demonstrate that in some perinuclear staining (P-ANCA) patients' sera autoantibodies against other myeloid lysosomal enzymes can be detected, such as antimyeloperoxidase and antielastase. C-ANCA and P-ANCA thus represent a novel class of autoantibodies directed against myeloid lysosomal enzymes. The originally described Wegener-specific C-ANCA show an apparently uniform specificity for the 29,000 serine protease. In contrast, P-ANCA may recognize myeloperoxidase as well as elastase and/or other antigens.

Topics: Antibodies, Monoclonal; Antigens; Autoantibodies; Carrier Proteins; Cathepsin G; Cathepsins; Cytochalasin B; Cytoplasm; Enzyme-Linked Immunosorbent Assay; Granulomatosis with Polyangiitis; Humans; Immunosorbent Techniques; Isoflurophate; Leukocyte Elastase; Lysosomes; Molecular Weight; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Pancreatic Elastase; Serine Endopeptidases