Cytosolic Internalization of Anti-DNA Antibodies by Human Monocytes Induces Production of Pro-inflammatory Cytokines Independently of the Tripartite Motif-Containing 21 (TRIM21)-Mediated Pathway

Abstract

Anti-DNA autoantibodies are a hallmark of systemic lupus erythematosus (SLE). A subset of anti-DNA IgG autoantibodies is cell-internalizable; thus they can enter living cells in the form of free IgG. However, the contribution made by the Fc region of internalized free-form IgG to the cytokine response has not been studied, despite the recent discovery of tripartite motif-containing 21 (TRIM21), a cytosolic Fc receptor involved in immune signaling. This study used an internalizable IgG anti-DNA antibody (3D8) to examine the cytokine responses of human monocytes to the Fc region of cytosolic free IgG. Internalization of 3D8 IgG and a 3D8 single-chain variable fragment-Fc (scFv-Fc) induced production of IL-8 and TNF-α via activation of NF-κB. By contrast, a 3D8 scFv (comprising variable domains alone) did not. This suggests Fc-dependent cytokine signaling. A 3D8 IgG-N434D mutant that is not recognized by TRIM21 induced greater production of cytokines than 3D8 IgG. Moreover the amounts of cytokines induced by 3D8 IgG in TRIM21-knockdown THP-1 cells were higher than those in control cells, indicating that cytokine signaling is not mediated by TRIM21. The results suggest the existence of a novel Fc-dependent signaling pathway that is activated upon internalization of IgG antibodies by human monocytes.

Antigen-binding affinity and thermostability of chimeric mouse-chicken IgY and mouse-human IgG antibodies with identical variable domains

“…HEK293F cells were cultured in serum-free FreeStyle 293 media (ThermoFisher Scientific; cat# 12338018) with 8% CO 2 and shaking at 130 rpm in the 37 °C incubator. FreeStyle 293-F cells (100 ml) at a density of 2 × 10 6 cells/ml were transfected with 200 μg of KV10 plasmid encoding an Ab gene using 400 μg of polyethylenimine (PEI) reagent with a molecular weight of ~25 kDa (Polyscience; cat# 23966-2), a final PEI concentration of 4 μg/ml 46 . After 7 days, the culture supernatants were harvested by centrifugation, and two chimeric MC-IgY and two chimeric MH-IgG proteins were purified by affinity chromatography using Protein L (GE Healthcare; cat# 17-5478-01) and Protein A column (GE Healthcare; cat# 17-1279-01), respectively.…”

An Emerging Role for Anti-DNA Antibodies in Systemic Lupus Erythematosus

“…Soon after, they reported similar findings with anti-DNA antibodies as well [13]. Initially, these findings met with skepticism, but gradually, many studies confirmed this phenomenon [14][15][16]. The mechanisms responsible for internalization are multifarious.…”