Figure 4.

Apoptotic fibroblast immune complexes containing Y RNA, Ro60 and anti-Ro60 stimulate a TLR7-dependent TNFα secretion by macrophages. PBMC-derived human macrophages (A) and PMA-stimulated THP-1 (B) secreted TNFα when co-cultured with wild-type (WT), FLAG₃-Ro60, or FLAG₃-Ro60(K170A R174A) apoptotic fibroblasts opsonized with anti-Ro60 (open bars) but not control IgG (shaded bars). Co-cultures with Ro60 knockout (KO) or FLAG₃-Ro60(H187S) fibroblasts, preincubated with anti-Ro60 Ab showed lower levels of TNFα that were equivalent to control IgG treatments. C) To support the role of the TLR pathway in macrophage activation, TNFα secretion by THP-1 stimulated by apoptotic fibroblast immune complexes containing Y RNA and anti-Ro60 Ab (open bars) was inhibited by IRS661 (shaded bars), a TLR7 antagonist. Values are mean ± SEM for 5 experiments. D) To support the association of macrophage activation with anti-Ro60 Ab, β₂GPI, previously shown to compete with anti-Ro60 Ab for binding to apoptotic cells, was added to wild-type apoptotic fibroblasts prior to opsonization with anti-Ro60 Ab and co-culture with THP-1. Consistent with the reported interaction of Ro60 and β₂GPI (25, 26); β₂GPI caused a dose-dependent inhibition of TNFα secretion. Values are mean ± SEM for 3 experiments. *P<0.05, ** P≤0.005.