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B lymphocytes infiltrating Wegener's granuloma: the immunoglobulin VH gene repertoire from granulomatous tissues displays an antigen-driven maturation and suggests a microbial trigger


In Wegener's granulomatosis (WG) the development of a generalized vasculitis is based on strong expression of proteinase 3 (PR3) on the surface of neutrophil granulocytes and the appearance of circulating high-affinity anti-PR3 autoantibodies (PR3-ANCAs) suggesting an antigen-driven B lymphocyte maturation.


In order to analyze mechanisms relevant to the induction of ANCA, we compared the immunoglobulin heavy chain (VH) gene repertoire in biopsy specimen of granulomas from two cases of ANCA-negative localized WG and from two cases of ANCA-positive generalized WG with a healthy control.


Frozen tissue samples were stained for B lymphocytes by anti-CD20 APAAP. DNA was prepared, purified and subjected to a PCR with individual VH primers matching to the families VH1–VH6 and a JH consensus primer. After bacterial subcloning PCR products were sequenced and characterized for gene usage and mutational pattern.


Immunohistology revealed lymphoid infiltrates within typical WG granulomas containing more than 30% B cells, which is suggestive of germinal center formation (Fig. 1). One hundred and fifty differently rearranged VH genes from WG tissues were characterized and compared with 84 VH genes from peripheral blood of a healthy donor. The mutation frequency as well as the ratio of amino acid replacement to silent mutations indicate an antigen-driven selection of antibodies within such germinal center-like regions of the granuloma in WG and differ significantly from the healthy control. The VH genes in WG revealed both striking similarities to published PR3-ANCA-encoding genes as well as to VH genes from Staphylococcus aureus superantigen-affine B cells. Furthermore, 50% of mutations within the binding-site coding regions led to negatively charged amino acids that favour affinity to the positively charged PR3.

Figure 1

CD20 staining of a cryosection from an endonasal Wegener granuloma.


In WG a subset of autoreactive PR3-producing B cells may be induced to produce PR3-ANCA within granulomatous lesions after Staphylococcus aureus B-cell superantigen stimulation. Selected B cells undergo affinity maturation and differentiate into plasma cells that produce high levels of circulating ANCAs, which are involved in the pathogenesis of the systemic vasculitis.


This work is supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 367, A11) and by the University of Luebeck (FUL N20-2001).

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  • Amino Acid Replacement
  • Gene Repertoire
  • Lymphoid Infiltrate
  • Germinal Center Formation
  • Lymphocyte Maturation