Anti-β2-glycoprotein I antibody with DNA binding activity enters living monocytes via cell surface DNA and induces tissue factor expression

Abstract

SummaryAutoantibodies characteristic for anti‐phospholipid syndrome (APS) and systemic lupus erythematosus (SLE) are anti‐β2‐glycoprotein I (β2GPI) antibodies and anti‐DNA antibodies, respectively, and almost half of APS cases occur in SLE. Anti‐β2GPI antibodies are recognized to play a pivotal role in inducing a prothrombotic state, but the precise mechanism has not been fully elucidated. In a widely accepted view, binding of anti‐β2GPI antibodies to cell surface β2GPI in monocytes and endothelial cells triggers the Toll‐like receptor 4‐myeloid differentiation primary response 88 (TLR)‐4‐MyD88) signaling pathway which leads to activation of p38 mitogen‐activated protein kinase (MAPK), mitogen‐activated protein kinase kinase 1/extracellular signal‐regulated kinases (MEK‐1/ERK) and/or nuclear factor kappa B (NF‐κB) and expression of tissue factor (TF). However, resting cells do not express substantial amounts of TLR‐4. Previously, we generated a mouse monoclonal anti‐β2GPI antibody WB‐6 and showed that it induced a prothrombotic state – including TF expression on circulating monocytes – in normal mice. In the current study, we aimed to clarify the mechanism of interaction between WB‐6 and resting monocytes, and found that WB‐6 exhibits binding activity to DNA and enters living monocytes or a monocytic cell line and, to a lesser extent, vascular endothelial cells. Treatment of the cells with DNase I reduced the internalization, suggesting the involvement of cell surface DNA in this phenomenon. Monocytes harboring internalized WB‐6 expressed TF and tumor necrosis factor (TNF)‐α which, in turn, stimulated endothelial cells to express intercellular adhesion molecule 1 (ICAM‐I) and vascular cell adhesion molecule 1 (VCAM‐I). These results suggest the possibility that a subset of anti‐β2GPI antibodies with dual reactivity to DNA possesses ability to stimulate DNA sensors in the cytoplasm, in addition to the cell surface receptor‐mediated pathways, leading to produce proinflammatory and prothrombotic states.

Monoclonal anti-dsDNA antibody 2C10 escorts DNA to intracellular DNA sensors in normal mononuclear cells and stimulates secretion of multiple cytokines implicated in lupus pathogenesis

“…As we have observed previously, anti-dsDNA antibody 2C10 enters live monocytes and the monocytic cell line THP-1 [16]. To confirm if the current batch of 2C10 actually enters live cells, we tested the effect of fixation and permeabilization (F/P) of THP-1 cells.…”

Anti-DNA antibodies cross-reactive with β2-glycoprotein I induce monocyte tissue factor through the TLR9 pathway

“…Previously, based on the results of direct binding ELISA, we had reported that the monoclonal antibody WB-6 purified using a Protein-G column binds to both DNA and CL-b 2 GPI [16]. Although this was reproducible, we were not fully confident of the reactivity because one partner was nucleic acid and the other protein; do they share a common structure?…”

“…WB-6 enters THP-1 cells, monocytes and endothelial cells [16]. In the present study, we investigated the kinetics of this phenomenon using PBMCs from healthy volunteers comparing WB-6 with other monoclonal anti-DNA antibodies.…”

“…Antibody binding to CL-b 2 GPI was determined by enzyme-linked immunosorbent assay (ELISA) as described previously [15]. DNA binding was also determined by ELISA as described [16]. Calf-thymus DNA (Sigma Aldrich) was dissolved in Tris-buffered saline (TBS; 25 mM Tris, 140 mM NaCl, pH 7.4) containing 5 mM EDTA, and used as doublestranded (ds) DNA.…”

“…Previously, we generated a mouse monoclonal antibody designated WB-6 from a lupus-prone male (NZW x BXSB)F1 mouse, and reported that it binds to CL-b 2 GPI, induces TF on normal human as well as mouse monocytes, and induces a prothrombotic state in an in vivo mouse model of APS [15]. During studies on how WB-6 interacts with monocytes, we found that this antibody binds to DNA as well as to CL-b 2 GPI, and enters living cells via binding to the cell surface DNA [16]. Based on these findings, we proposed a novel hypothesis that some anti-CL-b 2 -GPI antibodies are endocytosed into living monocytes together with the bound DNA, and thus stimulate intracellular DNA sensors, leading to expression of TF on the cells.…”