Artikel
The tissue-derived Ig Vh gene repertoire in granulomatosis with polyangiitis (GPA) reflects the development of autoreactivity
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Veröffentlicht: | 1. September 2015 |
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Introduction: GPA is characterised by necrotising granulomatosis and PR3-ANCA-associated vasculitis, predominantly of small blood vessels. Although much has been discovered about the vasculitic pathogenesis, there is still a lack of knowledge regarding the development and/or persistence of pathogenically relevant PR3-ANCA. We propose that the granulomatous inflammation in various diseased tissues provides structural elements which support the selection and maturation of autoantibody-producing cells.
Methods: Using improved analytical tools, for instance in quantifying selection (BASELINe test of significance for antigen-driven selection), functionally rearranged tissue-derived Ig Vh gene fragments (n=245) from GPA patients (n=14) were re-analysed, in comparison to Ig Vh genes from peripheral blood B cells (n=212) of healthy controls (HC, n=4). In addition, immunofluorescence (IF) stainings of GPA tissues were performed to search for PR3-ANCA-producing cells.
Results: With respect to Vh families, the tissue-derived Ig Vh gene fragments showed a higher proportion of Vh1 and Vh4 compared to B cells from peripheral blood of HC. The Vh4-34 gene segment represented about 8% of the tissue-derived Ig Vh genes in GPA. In terms of CDR3 length, the tissue-derived Ig Vh genes displayed less short (1-9 amino acids) and more prolonged CDR3’s (> than 15 amino acids) compared to B cells from peripheral blood of HC. Regarding selection, the re-analysis of Ig Vh gene fragments from whole GPA tissues yielded a significant evidence of negative selection against replacement mutations in the framework regions (p<0.005). This result was also true for Ig Vh gene fragments from single tissue-derived CD138+ cells of GPA patients. No clonal expansions were observed connecting two inflamed tissue samples in a GPA patient (nasal mucosa vs lung), but three clones each were detected, irrespective of the analysis of whole tissue or individual CD20+ cells. Further, IF stainings indicated that an idiotype of PR3-ANCA (5/7 Id) is binding to cells with a polymorphonuclear appearance, which partially fits to the hypothesis of the ANCA-cytokine sequence and is suggestive of PR3-ANCA presence in granulomatous inflammation.
Conclusion: Altogether, genetic and protein data support the assumption that in GPA the granulomatous inflammation is a place, where autoreactivity, eventually directed against PR3, develops and/or persists.