Background/Purpose: Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides (AAV) are characterized by the presence of circulating autoantibodies that are often directed against proteinase 3 (PR3). Although the mechanisms that lead to ANCA production in AAV are not clear, bacterial infections have been linked to disease development. We have reported that unmethylated CpG oligodeoxynucleotides (CpG-ODN), which resemble bacterial DNA, in combination with IL-2 enhance ANCA production in vitro. Recent studies have highlighted the role of IL-21 in plasma cell formation and antibody production by synergizing with B cell activating factor (BAFF). This study aimed to assess the involvement of CpG-ODN, IL-21, and BAFF in the mechanisms that contribute to ANCA production in AAV patients.
Methods: Twenty two patients with PR3-AAV (18 in clinical remission, 4 with active disease) and 8 healthy controls (HC) were included in the study. Peripheral blood mononuclear cells (PBMC) were isolated and cultured in vitro for 12 days in the presence of BAFF and IL-21, with or without CpG-ODN. IgG production was measured in culture supernatants by ELISA and PR3-ANCA production was quantified by Phadia EliA and expressed in response units (RU). The percentage of circulating IL-21 producing CD4+ T cells was analyzed by flow cytometry in blood samples stimulated ex vivo with phorbol-myristate-acetate and calcium ionophore in the presence of brefeldin A. CFSE Cell Proliferation Kit was used to assess the effect of BAFF, IL-21, and CpG-ODN on B cell proliferation.
Results: PBMC stimulation with CpG-ODN and IL-2 significantly increased in vitro production of IgG in both HC and patients (P=0,0004) whereas PR3-ANCA production was detected in patient samples only (RU median 0,85 (range 0,00-36,72), compared to 0,10 (0,00-0,14) in HC). Stimulation with BAFF and IL-21 also significantly increased IgG production in HC and patients (P=0,0001) and ANCA production in patient samples (median 0,90 (range 0,00-58,80) versus 0,11 (0,00-0,12) in HC), which could be further augmented by addition of CpG-ODN (median 1,06 (range 0,00-140,00) versus 0,17 (0,10-0,26) in HC). Compared to HC, the proportion of IL-21 producing CD4+ T cells was significantly increased in the circulation of AAV patients. Preliminary data indicate that stimulation with BAFF, IL-21 or the combination of BAFF/IL-21 does not induce B cell proliferation. In contrast, stimulation with CpG-ODN alone induced proliferation in 11,6% of B cells, whereas the combined treatment with BAFF, IL-21 and CpG-ODN induced proliferation in 57,8% of B cells.
Conclusion: IL-21, BAFF and CpG ODN synergize in promoting IgG and PR3-ANCA production from PBMCs of AAV patients in vitro. This effect was associated with substantial B cell proliferation. The increased percentage of IL-21 producing CD4+ T cells in AAV patients suggests the involvement of IL-21 in ANCA production in vivo. Overall, these data indicate that the interplay between endogenous B cell stimuli and bacterial products may contribute to PR3-ANCA reactivation in AAV via B cell activation and proliferation.
Disclosure:
N. Lepse,
None;
J. Land,
None;
A. Rutgers,
None;
C. G. M. Kallenberg,
None;
C. A. Stegeman,
None;
P. Heeringa,
None;
W. H. Abdulahad,
None.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/interleukin-21-b-cell-activating-factor-and-unmethylated-cpg-oligodeoxynucleotides-synergize-in-promoting-anti-proteinase-3-autoantibody-production-in-vitro/