Gastroenterology · 2000
"Atypical p-ANCA" in IBD and hepatobiliary disorders react with a 50-kilodalton nuclear envelope protein of neutrophils and myeloid cell lines.
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Terjung B, Spengler U, Sauerbruch T, Worman H J
Аннотация
BACKGROUND & AIMS: Atypical "antineutrophil cytoplasmic antibodies" (ANCA) are present in patients with ulcerative colitis (UC), primary sclerosing cholangitis (PSC), and autoimmune hepatitis (AIH). Recently, we showed that atypical p-ANCA react with nuclear envelope proteins of neutrophils. Based on this observation, we aimed to characterize the nuclear antigen recognized by atypical p-ANCA.
METHODS: We prepared cytoplasmic and nuclear extracts of human neutrophils, human HL-60, and murine 32D myeloid cells. Proteins were resolved by 1- and 2-dimensional gel electrophoresis. Reactive proteins were detected by immunoblotting with sera from 118 individuals (UC, 25; PSC, 28; AIH, 35; disease and normal controls, 30). Atypical p-ANCA (n = 64) were affinity-purified against the reactive protein and investigated for their immunofluorescence pattern using confocal microscopy.
RESULTS: Immunoblotting showed reactivity to a myeloid-specific 50-kilodalton nuclear protein with an isoelectric point of pH 6.0 detected in 92% (59 of 64) of the patients with inflammatory bowel or hepatobiliary diseases and atypical p-ANCA. Affinity-purified antibodies against the 50-kilodalton protein gave a nuclear rim-like fluorescence on myeloid cells examined by immunofluorescence microscopy. Affinity-purified antibodies did not recognize antigens in nonmyeloid cells.
CONCLUSIONS: Atypical p-ANCA in UC, PSC, or AIH recognize a 50-kilodalton myeloid-specific nuclear envelope protein.
METHODS: We prepared cytoplasmic and nuclear extracts of human neutrophils, human HL-60, and murine 32D myeloid cells. Proteins were resolved by 1- and 2-dimensional gel electrophoresis. Reactive proteins were detected by immunoblotting with sera from 118 individuals (UC, 25; PSC, 28; AIH, 35; disease and normal controls, 30). Atypical p-ANCA (n = 64) were affinity-purified against the reactive protein and investigated for their immunofluorescence pattern using confocal microscopy.
RESULTS: Immunoblotting showed reactivity to a myeloid-specific 50-kilodalton nuclear protein with an isoelectric point of pH 6.0 detected in 92% (59 of 64) of the patients with inflammatory bowel or hepatobiliary diseases and atypical p-ANCA. Affinity-purified antibodies against the 50-kilodalton protein gave a nuclear rim-like fluorescence on myeloid cells examined by immunofluorescence microscopy. Affinity-purified antibodies did not recognize antigens in nonmyeloid cells.
CONCLUSIONS: Atypical p-ANCA in UC, PSC, or AIH recognize a 50-kilodalton myeloid-specific nuclear envelope protein.
Предметные рубрики MeSH
AdolescentAdultAnimalsAntibodies, Antineutrophil CytoplasmicAntibody SpecificityCOS CellsCell FractionationChildCholangitis, SclerosingColitis, UlcerativeFemaleFluorescent Antibody TechniqueHL-60 CellsHeLa CellsHepatitis, AutoimmuneHumansImmune SeraLiverMaleMiddle AgedMolecular WeightNeutrophilsNuclear EnvelopeNuclear Proteins