Abstract
The inhibition of Fcγ receptors (FcγR) is an attractive strategy for treating diseases driven by IgG immune complexes (IC). Previously, we demonstrated that an engineered tri-valent arrangement of IgG1 Fc domains (SIF1) potently inhibited FcγR activation by IC, whereas a penta-valent Fc molecule (PentX) activated FcγR, potentially mimicking ICs and leading to Syk phosphorylation. Thus, a precise balance exists between the number of engaged FcγRs for inhibition versus activation. Here, we demonstrate that Fc valency differentially controls FcγR activation and inhibition within distinct subcellular compartments. Large Fc multimer clusters consisting of 5-50 Fc domains predominately recruited Syk-mScarlet to patches on the plasma membrane, whereas PentX exclusively recruited Syk-mScarlet to endosomes in human monocytic cell line (THP-1 cells). In contrast, SIF1, similar to monomeric Fc, spent longer periods docked to FcγRs on the plasma membrane and did not accumulate and recruit Syk-mScarlet within large endosomes. Single particle tracking (SPT) of fluorescent engineered Fc molecules and Syk-mScarlet at the plasma membrane imaged by total internal reflection fluorescence microscopy (SPT-TIRF), revealed that Syk-mScarlet sampled the plasma membrane was not recruited to FcγR docked with any of the engineered Fc molecules at the plasma membrane. Furthermore, the motions of FcγRs docked with recombinant Fc (rFc), SIF1 or PentX, displayed similar motions with D ~ 0.15 μm2/s, indicating that SIF1 and PentX did not induce reorganization or microclustering of FcγRs beyond the ligating valency. Multicolor SPT-TIRF and brightness analysis of docked rFc, SIF1 and PentX also indicated that FcγRs were not pre-assembled into clusters. Taken together, activation on the plasma membrane requires assembly of more than 5 FcγRs. Unlike rFc or SIF1, PentX accumulated Syk-mScarlet on endosomes indicating that the threshold for FcγR activation on endosomes is lower than on the plasma membrane. We conclude that the inhibitory effects of SIF1 are mediated by stabilizing a ligated and inactive FcγR on the plasma membrane. Thus, FcγR inhibition can be achieved by low valency ligation with SIF1 that behaves similarly to FcγR docked with monomeric IgG.
Highlights
Immune complexes (IC) containing immunoglobulin G (IgG) are a hallmark of many autoimmune diseases and inflammatory reactions
The engineered multimeric Fc molecules, rFc, SIF1, and pentameric Fc arranged in an X-geometry (PentX), provided new insight into the minimal number of Fcg receptors (FcgR) that must be engaged for signaling and that this threshold depends on the subcellular environment
Activation predict a minimal number of ligated and clustered receptors is necessary for activation, our findings give the surprising result that endosomes may afford a lower threshold of activation for the minimum number of FcgRs than the plasma membrane
Summary
Immune complexes (IC) containing immunoglobulin G (IgG) are a hallmark of many autoimmune diseases and inflammatory reactions. The activation of Fcg receptors (FcgR) on macrophages by IC-IgG contributes to cytokine signaling and inflammatory pathogenesis in autoimmunity including the recruitment of neutrophils, monocytes, T cells, natural killer cells (NK cells), and additional macrophages [1, 2]. In systemic lupus erythematosus (SLE), a chronic autoimmune disease, IC-IgG deposits in organs and tissues [3] and are recognized by FcgRs on macrophages, monocytes and neutrophils [4]. In SLE, macrophages are overwhelmed and unable to clear the ICs leading to continued macrophage activation, sustained secretion of pro-inflammatory cytokines, autoantibody production, tissue damage and acute phases of the disease [5]. Inhibiting FcgR activation and disrupting this inflammatory circuit is an attractive therapeutic strategy for treating autoimmunity [6]
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