cathepsin-g and Inflammatory-Bowel-Diseases

Atypical antineutrophil cytoplasm antibodies (A-ANCA) are defined here as ANCA detected by IIF and not directed against the predominant ANCA antigens, proteinase 3 (PR3) and myeloperoxidase (MPO). A-ANCA are found in a variety of clinical conditions, namely rheumatoid arthritis, inflammatory bowel diseases, chronic hepatic diseases and several infections including HIV infection. They are directed against a variety of still ill-defined neutrophil antigens and most frequently yield a perinuclear pattern (P-ANCA) of binding by indirect immunofluorescence on ethanol fixed neutrophils. This paper reviews the literature on A-ANCA and our recent data suggesting that, among others, cathepsin G is one of the predominant antigen targets of A-ANCA. From a clinical point of view, the distinction between MPO-ANCA and A-ANCA is not possible by indirect immunofluorescence (IIF). The determination of ANCA antigens by specific ELISA is therefore necessary to differentiate P-ANCA with MPO specificity from those with undefined specificity. This is of importance because the clinical value of MPO-ANCA is clearly established while the presence of A-ANCA is difficult to interpret given their occurrence in a large variety of clinical conditions.

Topics: Antibodies, Antineutrophil Cytoplasmic; Antibody Specificity; Arthritis, Rheumatoid; Autoantibodies; Autoantigens; Autoimmune Diseases; Cathepsin G; Cathepsins; Cytoplasm; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Humans; Infections; Inflammatory Bowel Diseases; Liver Diseases; Myeloblastin; Neutrophils; Pancreatic Elastase; Peroxidase; Serine Endopeptidases

The pathogenic importance of antineutrophil cytoplasmic antibodies (ANCAs) in inflammatory bowel disease (IBD) is unclear and target antigen localisation studies may lend insight to the specific pathogenic mechanisms of IBD. In this pilot study, we looked at occurrence of ANCA in Asian IBD patients. In ANCA-positive samples, we analysed for the presence of target antigens i.e. proteinase 3, lactoferrin, myeloperoxidase, elastase, cathepsin G and lysozyme.. This prospective study was carried out from July 1997 to February 1998. Sera were screened for ANCAs with indirect immunofluorescent test and tested with an enzyme immunoassay (ELISA) kit which provides a semi-quantitative assay for human IgG autoantibodies against 6 antigens: proteinase 3, lactoferrin, myeloperoxidase, elastase, cathepsin G and lysozyme.. A total of 75 patients were studied: 50 with IBD and 25 controls with functional bowel disease. Ten had Crohn's disease (CD) and 40 had ulcerative colitis (UC). There was no racial predilection among the Chinese, Malays or Indians. In CD, 1 was positive for cytoplasmic ANCA (cANCA) and 2 for perinuclear ANCA (pANCA). In UC, 4 were positive for pANCA, 15 for atypical perinuclear ANCA (apANCA) and 1 for cANCA. In the CD and UC population, the proportion positive for ANCA was 30% and 50%, respectively. There was no ANCA detected among the controls. Of those ANCA-positive IBD patients (n:23), only 1 demonstrated anti-myeloperoxidase antibodies. No antibodies were detected against the other 5 antigens tested.. This pilot Singapore study concludes that there is no significant ANCA association with proteinase 3, lactoferrin, myeloperoxidase, elastase, cathepsin G and lysozyme.

Topics: Adult; Aged; Antibodies, Antineutrophil Cytoplasmic; Biomarkers; Cathepsin G; Cathepsins; Enzyme-Linked Immunosorbent Assay; Female; Fluorescent Antibody Technique, Indirect; Humans; Inflammatory Bowel Diseases; Lactoferrin; Male; Middle Aged; Muramidase; Myeloblastin; Pancreatic Elastase; Peroxidase; Pilot Projects; Prognosis; Prospective Studies; Reference Values; Sensitivity and Specificity; Serine Endopeptidases; Singapore

While proteases are essential in gastrointestinal physiology, accumulating evidence indicates that dysregulated proteolysis plays a pivotal role in the pathophysiology of inflammatory bowel disease (IBD). Nonetheless, the identity of overactive proteases released by human colonic mucosa remains largely unknown. Studies of protease abundance have primarily investigated expression profiles, not taking into account their enzymatic activity. Herein we have used serine protease-targeted activity-based probes (ABPs) coupled with mass spectral analysis to identify active forms of proteases secreted by the colonic mucosa of healthy controls and IBD patients. Profiling of (Pro-Lys)-ABP bound proteases revealed that most of hyperactive proteases from IBD secretome are clustered at 28-kDa. We identified seven active proteases: the serine proteases cathepsin G, plasma kallikrein, plasmin, tryptase, chymotrypsin-like elastase 3 A, and thrombin and the aminopeptidase B. Only cathepsin G and thrombin were overactive in supernatants from IBD patient tissues compared to healthy controls. Gene expression analysis highlighted the transcription of genes encoding these proteases into intestinal mucosae. The functional ABP-targeted proteomic approach that we have used to identify active proteases in human colonic samples bears directly on the understanding of the role these enzymes may play in the pathophysiology of IBD.

Topics: Cathepsin G; Chromatography, Liquid; Gene Expression Profiling; Gene Expression Regulation; Humans; Inflammatory Bowel Diseases; Intestinal Mucosa; Proteomics; Serine Proteases; Tandem Mass Spectrometry; Thrombin; Up-Regulation

Neutrophil (PMN) infiltration and associated release of serine proteases contribute to epithelial injury during active phases of mucosal disorders such as inflammatory bowel disease. Previous studies have demonstrated that PMN contact with basolateral surfaces of intestinal epithelial cells in the presence of a chemoattractant results in disruption of barrier function even without transmigration. Similarly, serine protease-mediated activation of epithelial protease-activated receptors (PARs) has been shown to increase permeability. In this study, we assessed whether transmigrating PMNs can regulate barrier function through epithelial PAR activation. Transepithelial resistance (TER) decreased significantly after PMN contact with basolateral surfaces of T84 monolayers or after incubation with PMN elastase and proteinase-3, but not cathepsin G. Inhibition of PMN serine proteases, but not selective inhibition of elastase or cathepsin G, prevented the fall in TER induced by PMN contact and blocked PMN transepithelial migration. Basolateral, but not apical, PAR-1 and -2 activation with selective agonists also decreased TER. PAR-1 and -2 were localized intracellularly and in close proximity to lateral surfaces beneath tight junctions, and expression was increased in colonic mucosa from individuals with Crohn's disease. Combined, but not individual, transfection with small interfering RNAs targeted against epithelial PAR-1 and -2, prevented the fall in TER induced by PMN contact. Furthermore, basolateral PAR-1 and -2 activation induced phosphorylation of myosin L chain kinase and regulatory myosin L chain. Lastly, epithelial PAR-1 and -2 knockdown decreased the rate of PMN transepithelial migration. These results suggest that protease-mediated epithelial PAR-1 and -2 activation, by migrating PMNs, induces signaling events that increase epithelial permeability thereby facilitates PMN transepithelial migration.

Topics: Cathepsin G; Cathepsins; Cell Movement; Cells, Cultured; Chemotactic Factors; Coculture Techniques; Gene Expression Regulation; Humans; Inflammatory Bowel Diseases; Intestinal Mucosa; Myeloblastin; Neutrophil Infiltration; Neutrophils; Pancreatic Elastase; Permeability; Receptor, PAR-1; Receptor, PAR-2; Serine Endopeptidases; Signal Transduction; Substrate Specificity; Tight Junctions

Topics: Antibodies, Antineutrophil Cytoplasmic; Autoantibodies; Cathepsin G; Cathepsins; Humans; Inflammatory Bowel Diseases; Serine Endopeptidases

Our aim was to investigate the prevalence of antineutrophil cytoplasmic antibodies (ANCA) in Japanese patients with ulcerative colitis (UC) and Crohn's disease (CD), and the putative antigens recognized by perinuclear staining pattern ANCA (p-ANCA)-positive sera.. Sera from UC (n = 52) and CD (n = 43) patients, and from healthy controls (n = 74) were studied. The indirect immunofluorescence (IIF) method was used for the detection of ANCA and its binding pattern. p-ANCA-positive sera were studied further for putative antigens. ELISAs using lactoferrin (Lf), myeloperoxidase (MPO), and cathepsin G (Cat G) as antigens were performed.. ANCA was positive in 40 of the 52 (76.9%) UC (p-ANCA in 33) and in 32 of the 43 (74.4%) CD (p-ANCA in 31) patients. UC and CD patients showed significantly higher titers of p-ANCA than controls; however, no significant difference was observed between UC and CD. In UC, 23, 17, and nine of the 33 patients with p-ANCA-positive sera showed reactivity with Lf, MPO, and Cat-G, respectively. In CD, 21, 20, and 11 of the 31 patients with p-ANCA-positive sera showed reactivity with Lf, MPO, and Cat-G, respectively. Fourteen of the UC and six of the CD patients showed reactivity with two different antigens, and seven of the UC and 11 of the CD patients showed reactivity with all three antigens. The presence of anti-Lf and anti-MPO antibodies was further confirmed by Western blotting.. ANCA is useful in distinguishing patients with IBD from normal subjects but is not sufficient for the differential diagnosis of CD and UC. p-ANCA reactivity might be derived from the recognition of heterogeneous neutrophil-associated antigens.

Topics: Adult; Animals; Antibodies, Antineutrophil Cytoplasmic; Antigen-Antibody Reactions; Antigens; Binding, Competitive; Biomarkers; Cathepsin G; Cathepsins; Colitis, Ulcerative; Crohn Disease; Diagnosis, Differential; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique, Indirect; Humans; Immunoglobulin G; Inflammatory Bowel Diseases; Japan; Lactoferrin; Milk; Peroxidase; Serine Endopeptidases; Serum Albumin, Bovine

To characterize the antigen specificity of circulating anti-neutrophil cytoplasmic antibodies (ANCAs) in inflammatory bowel disease (IBD).. Analysis of the prevalence of circulating ANCAs in patients with ulcerative colitis and Crohn's disease, by both non-specific methods (immunofluorescence against fixed neutrophil leukocytes) and specific antigen techniques (against purified neutrophil leukocyte constituents).. Indirect immunofluorescence against fixed polymorphonuclear leukocytes, and solid-phase enzyme-linked immunosorbent assay (ELISA) against neutrophil constituents (alpha-granules, elastase, myeloperoxidase, cathepsin g, lysozyme and lactoferrin).. Although results using immunofluorescence were typical of other studies (ulcerative colitis positive in 41%, Crohn's disease in 10%), ELISA studies showed antibody activity against neutrophil components in 69% of patients with ulcerative colitis and 39% of those with Crohn's disease. Antibodies in ulcerative colitis were commonly directed (in descending order) against lysozyme, cathepsin G, elastase, and lactoferrin, and in Crohn's disease against lysozyme.. Correlation of indirect immunofluorescence data and ELISA results indicated that even this large panel of specific antigens fails to identify all the ANCA targets in IBD. The lack of correlation between the findings of ANCAs, either in general or versus a specific target, and disease extent or activity in ulcerative colitis supports the suggestion that ANCAs are unlikely to be of primary importance in pathogenesis.

Topics: Adult; Aged; Antibodies; Antibodies, Antineutrophil Cytoplasmic; Autoantibodies; Cathepsin G; Cathepsins; Cholangitis, Sclerosing; Colitis, Ulcerative; Crohn Disease; Cytoplasmic Granules; Enzyme-Linked Immunosorbent Assay; Epitopes; Female; Fluorescent Antibody Technique, Indirect; Humans; Immunoglobulin G; Inflammatory Bowel Diseases; Lactoferrin; Leukocyte Elastase; Male; Middle Aged; Muramidase; Neutrophils; Pancreatic Elastase; Peroxidase; Serine Endopeptidases; Vasculitis

Anti-cathepsin G antibodies have been detected by using three different methods. i) Binding to azurophilic granules constituents after separation of purified alpha-granules on Matrex gel Orange A chromatography according to Kao. ii) Binding to azurophilic granules freezed and thawed after coating on ELISA plates. iii) Binding to purified cathepsin G in ELISA assay. Anti-cathepsin G antibodies were observed patient's sera with ulcerative colitis, primary sclerosing cholangitis, primary biliary cirrhosis and autoimmune hepatitis but not in controls or patients with chronic viral hepatitis or vasculitis.

Topics: Antibodies, Antineutrophil Cytoplasmic; Autoantibodies; Autoimmune Diseases; Cathepsin G; Cathepsins; Chromatography, Affinity; Cytoplasmic Granules; Enzyme-Linked Immunosorbent Assay; Freezing; Hepatitis; Hot Temperature; Humans; Immunoglobulin G; Inflammatory Bowel Diseases; Serine Endopeptidases