The relation between interleukin-10 and in vitro PR3-ANCA production

Objectives: Granulomatosis with polyangiitis (GPA) is associated with the presence of anti-neutrophil cytoplasmic autoantibodies (ANCA), mainly directed at proteinase-3 (PR3). Production of ANCA has been related to the occurrence of relapse. However, the mechanisms and factors that trigger PR3-ANCA production are unclear. Here, we examined the involvement of cytokine production in inducing or suppressing PR3-ANCA production. Methods: Peripheral blood mononuclear cells (PBMC) from 84 PR3-positive GPA patients and 24 healthy controls were cultured for 12 days in presence of CpG-oligodeoxynucleotides, Bcell activating factor and interleukin(IL)-21. Levels of PR3-ANCA IgG in culture supernatants were quantified by Phadia EliA and expressed in response units (RU). For 11 known ANCA producers the in vitro ANCA culture was repeated from cryopreserved PBMC, and IL10 was added to the culture. In addition, from a subset of patients 29 cytokines were measured in the supernatants by multiplex Luminex. For some cytokines this analysis was expanded by ELISA. Results: Sixty-five percent of patients were positive for in vitro PR3-ANCA production. To pinpoint differences in cytokine production, the 15 highest individual ANCA producers were matched with 15 non-producers and controls for Luminex analysis. Compared to patients that did not show in vitro ANCA production, those that did showed higher levels of several pro-inflammatory cytokines, most notably IL6, and higher levels of the regulatory cytokine IL10 in the supernatants. When the data-set was expanded by ELISA, no significant difference was observed for IL6, but patients with higher ANCA production showed significantly higher IL10 levels (P = 0.04). Also, when patients showed an increase of in vitro ANCA over time, a simultaneous increase of IL10 was seen. When exogenous IL10 was added to the in vitro ANCA assay this resulted in a significant decrease of PR3-ANCA when added at the start of the culture (figure 1, P = 0.005). When IL10 was added after six days of culture no significant effect on PR3-ANCA levels was observed. Conclusions: We demonstrate significantly higher levels of the regulatory cytokine IL10 in supernatants from patients that show high in vitro PR3-ANCA production. While these levels were not dissimilar from those found in healthy controls, they are not successful at suppressing the ANCA production in vitro. Exogenously added IL10 was shown capable of suppressing production of PR3-ANCA, at a higher concentration than found in the culture supernatants. This effect seems dependent on the timing of adding IL10.