OBJECTIVE Peripheral blood cells (PBMCs) from some patients with systemic sclerosis (SSc) express an interferon-alpha (IFNalpha) signature. The aim of this study was to determine whether SSc patient sera could induce IFNalpha and whether IFNalpha induction was associated with specific autoantibodies and/or clinical features of the disease. METHODS SSc sera containing autoantibodies against either topoisomerase I (anti-topo I; n = 12), nucleolar protein (ANoA; n = 12), or centromeric protein (ACA; n = 13) were cultured with a HeLa nuclear extract and normal PBMCs. In some experiments, different cell extracts or inhibitors of plasmacytoid dendritic cell (DC) activation, Fcgamma receptor II (FcgammaRII), endocytosis, or nucleases were used. IFNalpha was measured by enzyme-linked immunosorbent assay. RESULTS Topo I-containing sera induced significantly higher levels of IFNalpha as compared with all other groups. IFNalpha induction was inhibited by anti-blood dendritic cell antigen 2 (90%), anti-CD32 (76%), bafilomycin (99%), and RNase (82%). In contrast, ACAs induced low levels of IFNalpha even when necrotic, apoptotic, or demethylated extracts were used, despite the fact that CENP-B-binding oligonucleotide containing 2 CpG motifs effectively stimulated IFNalpha. IFNalpha production was significantly higher in patients with diffuse SSc (mean +/- SEM 641 +/- 174 pg/ml) than in those with limited SSc (215 +/- 66 pg/ml) as well as in patients with lung fibrosis than in those without. CONCLUSION Autoantibody subsets in SSc sera differentially induce IFNalpha and may explain the IFNalpha signature observed in SSc. IFNalpha is induced by plasmacytoid DCs and required uptake of immune complexes through FcgammaRII, endosomal transport, and the presence of RNA, presumably for interaction with Toll-like receptor 7. The higher IFNalpha induction in sera from patients with diffuse SSc than in those with limited SSc as well as in sera from patients with lung fibrosis suggests that IFNalpha may contribute to tissue injury.