Toll-like receptor 9 activation enhances B cell activating factor and interleukin-21 induced anti-proteinase 3 autoantibody production in vitro

Rheumatology (Oxford). 2016 Jan;55(1):162-72. doi: 10.1093/rheumatology/kev293. Epub 2015 Aug 28.


Objectives: Granulomatosis with polyangiitis (GPA) is a relapsing small-vessel vasculitis characterized by circulating ANCA against PR3. The mechanisms that trigger PR3-ANCA production are unknown. The aim of this study was to determine whether endogenous factors [B cell activating factor (BAFF) and IL-21] and exogenous factors [oligodeoxynucleotides containing CpG motifs (CpG-ODN)] synergize in stimulating PR3-ANCA production in GPA patients.

Methods: Peripheral blood mononuclear cells from GPA patients and healthy controls (HCs) were cultured in the presence of BAFF and IL-21, with or without CpG-ODN, for 12 days. PR3-ANCA production in culture supernatants was quantified by Phadia EliA. Phenotypic characterization and the influence of CpG-ODN treatment on IL-21 receptor (IL-21R), transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) and BAFF receptor (BAFF-R) expression on B cells was analysed by flow cytometry.

Results: Stimulation with BAFF and IL-21 significantly increased ANCA production in patient samples, which could be augmented further by addition of CpG-ODN. Stimulation with CpG-ODN increased the percentage of IL-21R(+) and TACI(+) B cells but did not affect BAFF-R expression. GPA patients had an increased percentage of circulating IL-21R(+) and a decreased percentage of TACI(+) circulating memory B cells when compared with HCs. Additionally, patients had decreased expression of BAFF-R on B cells, which was inversely correlated with BAFF concentrations in plasma.

Conclusion: Our data demonstrate that endogenous and exogenous factors can synergize to promote PR3-ANCA production. Mechanistically, CpG-ODN up-regulated IL-21R and TACI expression on B cells, possibly sensitizing these cells for IL-21- and BAFF-mediated signals. Agents inhibiting Toll-like receptor 9, BAFF and IL-21 signalling pathways may serve as potential therapeutics for intervention in GPA patients.

Keywords: ANCA; B cells; BAFF; BAFF-R; GPA; IL-21; IL-21R; TACI; TLR9; vasculitis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aged
  • Autoantibodies / immunology*
  • B-Cell Activating Factor / metabolism*
  • B-Lymphocytes / immunology
  • B-Lymphocytes / metabolism
  • B-Lymphocytes / pathology
  • Cell Proliferation
  • Enzyme-Linked Immunosorbent Assay
  • Female
  • Flow Cytometry
  • Granulomatosis with Polyangiitis / immunology*
  • Granulomatosis with Polyangiitis / metabolism
  • Granulomatosis with Polyangiitis / pathology
  • Humans
  • Interleukins / metabolism*
  • Lymphocyte Activation / immunology*
  • Male
  • Middle Aged
  • Myeloblastin / immunology*
  • Myeloblastin / metabolism
  • Toll-Like Receptor 9 / immunology
  • Toll-Like Receptor 9 / metabolism*


  • Autoantibodies
  • B-Cell Activating Factor
  • Interleukins
  • TLR9 protein, human
  • Toll-Like Receptor 9
  • Myeloblastin
  • interleukin-21