cathepsin-g and Colitis--Ulcerative

Cathepsin G (Cat-G) is a neutrophil serine-protease found in the colonic lumen of ulcerative colitis (UC) patients. Cat-G is able to activate protease-activated receptor-4 (PAR(4) ) located at the apical side of enterocytes, leading to epithelial barrier disruption. However, the mechanisms through which Cat-G triggers inflammation are not fully elucidated. The aims of our study were to evaluate in vivo the effects of UC fecal supernatants and Cat-G on epithelial barrier function and inflammation, and the connection between these two parameters.. Male balb/c mice were used in this study. We evaluated the effect of a 2-hour intracolonic infusion of 1) fecal supernatants from UC patients pretreated or not with specific Cat-G inhibitor (SCGI); 2) PAR(4) -activating peptide (PAR(4) -AP); and 3) Cat-G on colonic myeloperoxidase (MPO) activity and paracellular permeability (CPP). The involvement of PAR(4) was assessed by pretreating animals with pepducin P4pal-10, which blocks PAR(4) signaling. We investigated the role of myosin light chain (MLC) kinase by using its inhibitor, ML-7, and we determined phosphorylated MLC (pMLC) levels in mice colonic mucosa.. UC fecal supernatants, Cat-G, and PAR(4) agonist increased both CPP and MPO activity in comparison with healthy subjects fecal supernatants. ML-7 inhibited the CPP increase triggered by Cat-G by 92.3%, and the enhanced MPO activity by 43.8%. Intracolonic infusion of UC fecal supernatant determined an increased phosphorylation level of MLC.. These observations support that luminal factors such as Cat-G play an important proinflammatory role in the pathogenesis of colitis, mainly depending on CPP increase by MLC phosphorylation.

Topics: Administration, Rectal; Adolescent; Adult; Aged; Animals; Blotting, Western; Cathepsin G; Cell Membrane Permeability; Colitis; Colitis, Ulcerative; Colon; Feces; Humans; Intestinal Mucosa; Male; Mice; Mice, Inbred BALB C; Middle Aged; Peroxidase; Receptors, Thrombin; Young Adult

Impairment of the colonic epithelial barrier and neutrophil infiltration are common features of inflammatory bowel disease. Luminal proteases affect colonic permeability through protease-activated receptors (PARs). We evaluated: (i) whether fecal supernatants from patients with ulcerative colitis (UC) trigger alterations of colonic paracellular permeability and inflammation, and (ii) the roles of cathepsin G (Cat-G), a neutrophil serine protease, and its selective receptor, PAR(4), in these processes. Expression levels of both PAR(4) and Cat-G were determined in colonic biopsies from UC and healthy subjects. The effects of UC fecal supernatants on colonic paracellular permeability were measured in murine colonic strips. Involvement of Cat-G and PAR(4) was evaluated using pepducin P4pal-10 and specific Cat-G inhibitor (SCGI), respectively. In addition, the effect of PAR(4)-activating peptide was assessed. UC fecal supernatants, either untreated or pretreated with SCGI, were infused into mice, and myeloperoxidase activity was determined. PAR(4) was found to be overexpressed in UC colonic biopsies. Increased colonic paracellular permeability that was triggered by UC fecal supernatants was blocked by both SCGI (77%) and P4pal-10 (85%). Intracolonic infusion of UC fecal supernatants into mice increased myeloperoxidase activity. This effect was abolished by SCGI. These observations support that both Cat-G and PAR(4) play key roles in generating and/or amplifying relapses in UC and provide a rationale for the development of new therapeutic agents in the treatment of this disease.

Topics: Adult; Aged; Animals; Blotting, Western; Cathepsin G; Cathepsins; Cell Membrane Permeability; Colitis, Ulcerative; Feces; Humans; Immunohistochemistry; Intestinal Mucosa; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Middle Aged; Receptors, Thrombin; Reverse Transcriptase Polymerase Chain Reaction; Serine Endopeptidases

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

Topics: Antibodies, Antineutrophil Cytoplasmic; Cathepsin G; Cathepsins; Colitis, Ulcerative; Humans; Serine Endopeptidases

The presence of perinuclear anti-neutrophil cytoplasmic antibodies (P-ANCAs) and that of antibodies against cathepsin G, a target antigen for P-ANCAs, was determined in the sera of patients with ulcerative colitis (UC), relative to the endoscopic severity and disease activity. P-ANCAs were detected by indirect immunofluorescent assay (IIF) on ethanol-fixed human neutrophils. Antibodies to cathepsin G were detected by an enzyme-linked immunosorbent assay (ELISA) and Western blotting. P-ANCAs were detected by IIF in 62.5% of 32 patients with active UC. Anti-cathepsin G antibodies were detected in 40.6% of 32 patients with active UC, and their prevalence was significantly higher in patients with severe colitis, as determined by endoscopy, than in those with mild or moderate colitis (P < 0.05). The prevalence and titers of anti-cathepsin G antibodies were significantly higher during the active than the inactive phase of the disease (P < 0.05). Measurement of titers of anti-cathepsin G antibodies by ELISA in the serum is useful for evaluating the activity of UC.

Topics: Adolescent; Adult; Aged; Antibodies, Antineutrophil Cytoplasmic; Autoantibodies; Blotting, Western; Cathepsin G; Cathepsins; Colitis, Ulcerative; Enzyme-Linked Immunosorbent Assay; Female; Fluorescent Antibody Technique, Indirect; Humans; Male; Middle Aged; Prevalence; Serine Endopeptidases; Severity of Illness Index

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

In a previous study, we reported that the high mobility group (HMG) non-histone chromosomal proteins HMG1 and HMG2 were novel target antigens of P-ANCA. In this study, we determined the immunodiagnostic value of anti-HMG1/HMG2 antibodies in patients with UC. Sixty sera from patients with UC were tested for reactivity with HMG1 and HMG2 by means of ELISA. Anti-HMG1 antibody was detected in 32% of patients (40% of P-ANCA+ patients). Anti-HMG2 antibody was detected in 33% (40% of P-ANCA+ patients). Thirty-five percent of sera were positive for antibody to either HMG1 or HMG2 (43% of P-ANCA+ patients). P-ANCA+ patients expressed anti-HMG1/HMG2 antibodies with significantly greater frequency compared with P-ANCA- patients. Furthermore, the anti-HMG1/HMG2 antibodies were significantly related to disease activity in UC. Sixteen of the 18 UC patients, who had high titres of anti-HMG1 or -HMG2 antibody during the active phase, showed lower titres in the inactive phase. Anti-HMG1/HMG2 antibodies appear to be useful as a marker for disease activity in UC.

Topics: Adolescent; Adult; Aged; Animals; Antibodies; Antibodies, Antineutrophil Cytoplasmic; Blotting, Western; Cathepsin G; Cathepsins; Child; Colitis, Ulcerative; Enzyme-Linked Immunosorbent Assay; Female; High Mobility Group Proteins; Humans; Lactoferrin; Male; Middle Aged; Serine Endopeptidases; Swine

We examined the effect of ulinastatin in comparison with prednisolone (PSL) and salazosulfapyridine (SASP), well-known drugs for ulcerative colitis (UC), on two experimental UC models induced by dextran sulfate sodium (DSS) or trinitrobenzene sulfonic acid (TNB) in rats. Ulinastatin at the doses of 3000 approximately 10,000 units/kg/day (i.v.) significantly ameliorated the formation of erosion and infiltration of inflammatory cells in colonic mucosa in DSS-induced rat UC models. Moreover, ulinastatin at the dose of 10,000 units/kg/day (i.v.) significantly suppressed inflammation with ulcer in the colonic mucosa in TNB-induced rat UC models. PSL at the dose of 1 mg/kg/day (p.o.) also was as effective as ulinastatin on the above two UC models, while SASP at the dose of 100 mg/kg/day (p.o.) was less effective than ulinastatin and PSL. In addition, ulinastatin inhibited the activities of elastase and cathepsin G from human leukocytes, and it suppressed TNF alpha, IL-8 and superoxide production by rat macrophages and rabbit leukocytes in vitro. These results suggest that the suppression of inflammatory mediators produced by leukocytes is involved in the mechanism of the anti-UC action of ulinastatin.

Topics: Animals; Cathepsin G; Cathepsins; Colitis, Ulcerative; Female; Glycoproteins; Humans; Interleukin-8; Leukocyte Elastase; Leukocytes; Macrophages, Peritoneal; Male; Prednisolone; Rabbits; Rats; Rats, Sprague-Dawley; Rats, Wistar; Serine Endopeptidases; Sulfasalazine; Trypsin Inhibitors; Tumor Necrosis Factor-alpha

Bactericidal/permeability-increasing protein (BPI), a constituent of primary neutrophil granules, is a potent natural antibiotic and an antineutrophil cytoplasm antibody (ANCA) antigen in cases of vasculitis in which the target antigen is neither myeloperoxidase (MPO) nor proteinase-3 (PR3).. To investigate BPI as a possible target antigen for ANCAs in inflammatory bowel disease.. ANCAs were detected by routine immunofluorescence (IIF) and solid phase enzyme linked immunosorbent assay (ELISA) performed for antibodies to the purified neutrophil granule proteins; MPO, PR3, cathepsin-G, lactoferrin, and BPI in serum samples from 88 patients with inflammatory bowel disease (36 with Crohn's disease, 52 with ulcerative colitis). Thirty patients with bacterial enteritis acted as controls.. Significantly more patients with ulcerative colitis were ANCA positive by IIF (60%) than patients with Crohn's disease (28%) or infectious enteritis (23%) (p < 0.001). IgG anti-BPI antibodies were present in 29% of patients with ulcerative colitis, 14% of patients with Crohn's disease, and 23% of patients with infectious enteritis, occurring in 44% of those patients with inflammatory bowel disease who were ANCA positive by IIF. Antibodies to other ANCA antigens were rare. The presence of ANCAs was not related to either disease activity or extent; presence of anti-BPI antibodies was significantly related to both a lower serum albumin concentration (p = 0.001) and a higher erythrocyte sedimentation rate (p = 0.02) in patients with ulcerative colitis, and to colonic involvement in patients with Crohn's disease (p = 0.01).. BPI is a significant minority target antigen for ANCAs in inflammatory bowel disease that seems related to colonic Crohn's disease and disease activity in ulcerative colitis. Anti-BPI antibodies occur in infectious enteritis.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Antineutrophil Cytoplasmic; Antimicrobial Cationic Peptides; Blood Proteins; Case-Control Studies; Cathepsin G; Cathepsins; Colitis, Ulcerative; Crohn Disease; Enzyme-Linked Immunosorbent Assay; Female; Fluorescent Antibody Technique, Indirect; Humans; Immunoglobulin G; Lactoferrin; Male; Membrane Proteins; Middle Aged; Peroxidase; Prospective Studies; Serine Endopeptidases

We analysed the clinical significance of ANCA in patients with ulcerative colitis. On either an indirect immunofluorescence assay or an ELISA with fixed neutrophils, 71% (25/35) of the patients were positive for ANCA. However, only half of them reacted with either cathepsin G or lactoferrin. Western blot assays revealed positive bands in 40% (10/25) of the antibody-positive patients. The sizes of the bands detected were approximately 58, 47, 44, 40, and 28 kD. No significant correlation was found between the ANCA positivity and various variables, i.e. disease activity, extent of lesion, or treatment of the disease. The anti-cathepsin G and 28-kD positivity, however, significantly correlated with a refractory type of ulcerative colitis.

Topics: Adult; Aged; Antibodies, Antineutrophil Cytoplasmic; Autoantibodies; Blotting, Western; Cathepsin G; Cathepsins; Colitis, Ulcerative; Enzyme-Linked Immunosorbent Assay; Female; Humans; Lactoferrin; Male; Middle Aged; Molecular Weight; Neutrophils; Peroxidase; Serine Endopeptidases

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

To study ulcerative colitis associated neutrophil cytoplasmic antibodies (p-ANCA) in respect of class and subclass distribution, antigen specificity, and (sub)cellular localisation of the antigen(s) to which these antibodies are directed.. p-ANCA positivity was determined using the standard indirect immunofluorescence test (IIFT). The immunoglobulin (Ig) subclass distribution of p-ANCA was investigated using monoclonal antibodies directed against IgG1, IgG2, IgG3, and IgG4. Intracellular antigen localisation studies were performed on (fractionated) neutrophils using antigen-specific antibodies.. In contrast to vasculitis associated ANCA, ulcerative colitis p-ANCA are mainly of IgG1 and IgG3 subclass and lack IgG4. Ulcerative colitis p-ANCA are myeloid specific. IIFT data indicate that the related antigen(s) seem(s) to be located not in the cytosol, but in the granules (most likely the azurophil granules) of the neutrophil.. p-ANCA in ulcerative colitis have a different immunoglobulin subclass distribution than the ANCA of systemic necrotising vasculitis and necrotising and crescentic glomerulonephritis. This may point to differences in immune regulation between these diseases. Both cathepsin G and lactoferrin are recognised by a subpopulation of ulcerative colitis p-ANCA. In our series, eight out of 36 (22%) of ulcerative colitis associated p-ANCA react with lactoferrin and seven (19.5%) other sera with cathepsin G. None of them recognised both antigens. The main target antigen(s) of ulcerative colitis p-ANCA still remain(s) to be identified.

Topics: Antibodies, Antineutrophil Cytoplasmic; Autoantibodies; Biomarkers; Cathepsin G; Cathepsins; Colitis, Ulcerative; Cytoplasmic Granules; Fluorescent Antibody Technique; Humans; Immunoglobulin G; Lactoferrin; Monocytes; Neutrophils; Serine Endopeptidases

Autoantibodies directed against polymorphonuclear neutrophils (PMN) have been observed in serum from patients with ulcerative colitis (UC), Crohn's disease (CD) and primary sclerosing cholangitis (PSC) using indirect immunofluorescence and fixed granulocyte ELISA. Our study demonstrates the presence in the serum of these patients of autoantibodies which bind to an azurophilic granule component distinct from proteinase 3, elastase and myeloperoxidase. These autoantibodies thus belong to the ANCA family, but their antigen specificity differs from the already characterized ANCA antigens. We have found that the same ANCA antigen target, named UC-antigen, was recognized by serum IgG from patients with UC, CD and PSC. It was purified by Matrex Gel Orange A dye affinity chromatography and subsequent immunoabsorption of contaminant proteinase 3 with immobilized anti-proteinase 3 MoAb. The identity between this UC antigen and cathepsin G was demonstrated by their coelution from Matrex Gel Orange A column and the parallel titration of cathepsin G-specific enzymatic activity and UC-ANCA binding, both in partially purified UC antigen and in highly pure cathepsin G. Furthermore, the use of cathepsin G ELISA confirmed that UC, CD and PSC patients' IgG did indeed bind to cathepsin G. Comparison of the results obtained with azurophilic granule- and cathepsin G-ELISA as well as inhibition of ANCA binding by anti-cathepsin G polyclonal antibodies, revealed that in some patients cathepsin G is the main azurophilic granule target of ANCA while others have other ANCA specificities. The fact that UC, CD and PSC are frequently associated with cathepsin G ANCA, while rarely occurring in other types of vasculitis, is intriguing but suggests that these diseases may have a common pathogenetic mechanism.

Topics: Autoantibodies; Cathepsin G; Cathepsins; Cholangitis, Sclerosing; Colitis, Ulcerative; Crohn Disease; Cytoplasmic Granules; Humans; Lactoferrin; Myeloblastin; Neutrophils; Pancreatic Elastase; Peroxidase; Serine Endopeptidases