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

Background-Cell death bg: 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 greater than or equal to 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 non-apoptotic neutrophils were observed with respect to cellular ulcerative colitis associated ANCA distribution: (1) diffuse 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 diffuse 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.