Antineutrophil antibodies associated with ulcerative colitis interact with the antigen(s) during the process of apoptosis

Abstract

Background-Cell death by apoptosis seems to be an important mechanism for translocation to the cell surface of a variety of intracellular components capable of inducing autoantibody production. Aims-To identify the cellular location of antigen (Ag)-antineutrophil cytoplasmic antibodies (ANCA) in non-apoptotic human neutrophils, and to assess if ANCA associated with ulcerative colitis reacts with neutrophil antigen(s) during neutrophil apoptosis. The cellular distribution of Ag-ANCA in apoptotic neutrophils was also investigated. Methods-Sera from 18 ulcerative colitis patients known to be positive for perinuclear IgG-ANCA (titre >1/320), as assessed by indirect immunofluorescence (IIF), were analysed by immunofluorescent confocal laser scanning microscopy. ANCA were identified with fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) in non-apoptotic and apoptotic neutrophils, respectively. Apoptotic and non-apoptotic DNA was labelled with FITC and propidium iodide, respectively. Cycloheximide was added to polymorphonuclear leucocyte culture to induce apoptosis. Results-Three patterns of scanning laser immunofluorescence microscopy in nonapoptotic neutrophils were observed with respect to cellular ulcerative colitis associated ANCA distribution: (1) diVuse nuclear localisation (16.7%); (2) nuclear localisation in the nuclear periphery (50%); and (3) mixed nuclear and cytoplasmic localisation (33.4%). In all sera ANCA fluorescence colocalised almost completely with apoptotic DNA, with persistence of a diVuse and intense fluorescence. No significant changes in ANCA titres were found in non-apoptotic neutrophils. Conclusions-The antigen(s) of ANCA associated with ulcerative colitis seems to be localised in most cases in the neutrophil nucleus. The almost identical colocalisation of ANCA and apoptotic cleaved DNA suggests that intracellular DNA redistribution during neutrophil apoptosis may play a role in antigen exposure to the immune system and ANCA production in ulcerative colitis. (Gut 2000;47:74-78)

Autoantibodies pANCA, GAB and PAB in inflammatory bowel disease: prevalence, characteristics and diagnostic value

“…Th ese results are in agreement with other recent investigations, which strongly suggest that diff erent autoantigens are responsible for the pANCA immune response in patients with IBD. Th ese autoantigens are thought to be located not only in the cytoplasm of the granulocyte but also inside the granulocyte nucleus (histone 1, nonhistone chromosomal protein, high mobility group) [17,[27][28][29][30][31], with exhibited sensitivity on DNA-se treatment [17,27]. In agreement with the fi ndings of some other investigators [32][33][34], we found a positive reaction with cathepsin-G in two cases of UC; however, this specifi city was not signifi cantly diff erent from the control group, where two samples also tested positive with cathepsin-G.…”

Dynamin-Like Protein 1 Reduction Underlies Mitochondrial Morphology and Distribution Abnormalities in Fibroblasts from Sporadic Alzheimer's Disease Patients

“…33 Synchronization was confirmed by staining fibroblasts with propridium iodide/ RNase staining buffer (BD) 24 hours or 72 hours after serum addition or drug removal, which was examined by confocal microscopy. 34 To measure mitochondria distribution in synchronized fibroblasts, fibroblasts were plated on chamber slides after serum starvation or drug treatment for 24 hours in regular minimum essential medium supplemented with 10% fetal bovine serum and then transfected with mito-DsRed2. 48 hours after transfection, cells were fixed, immunostained, and examined as described below.…”

IgA class antineutrophil cytoplasmic antibodies in primary sclerosing cholangitis and autoimmune hepatitis

“…For IgG class ANCA, several proteins of the cytosol (actin, catalase, enolase) and the azurophilic (bactericidal/permeability increasing protein, cathepsin G) or specific granules (lactoferrin) have been suggested as target antigens [29,32]. Recent studies described nuclear proteins, particularly of the nuclear periphery, as candidate proteins of ‘atypical’ p‐ANCA [33–36]. Using enzyme‐linked immunosorbent assays, we could not demonstrate any reactivity of IgA class ANCA with the aforementioned cytosolic and granular proteins (data not shown).…”