Toll-like Receptor 4 Ligands Down-regulate Fcγ Receptor IIb (FcγRIIb) via MARCH3 Protein-mediated Ubiquitination

Abstract

Monocytes and macrophages are critical for the effectiveness of monoclonal antibody therapy. Responses to antibody-coated tumor cells are largely mediated by Fc␥ receptors (Fc␥Rs), which become activated upon binding to immune complexes. Fc␥RIIb is an inhibitory Fc␥R that negatively regulates these responses, and it is expressed on monocytes and macrophages. Therefore, deletion or down-regulation of this receptor may substantially enhance therapeutic outcomes. Here we screened a panel of Toll-like receptor (TLR) agonists and found that those selective for TLR4 and TLR8 could significantly down-regulate the expression of Fc␥RIIb. Upon further examination, we found that treatment of monocytes with TLR4 agonists could lead to the ubiquitination of Fc␥RIIb protein. A search of our earlier microarray database of monocytes activated with the TLR7/8 agonist R-848 (in which Fc␥RIIb was down-regulated) revealed an up-regulation of membrane-associated ring finger (C3HC4) 3 (MARCH3), an E3 ubiquitin ligase. Therefore, we tested whether LPS treatment could up-regulate MARCH3 in monocytes and whether this E3 ligase was involved with LPS-mediated Fc␥RIIb down-regulation. The results showed that LPS activation of TLR4 significantly increased MARCH3 expression and that siRNA against MARCH3 prevented the decrease in Fc␥RIIb following LPS treatment. These data suggest that activation of TLR4 on monocytes can induce a rapid down-regulation of Fc␥RIIb protein and that this involves ubiquitination.Monocytes and macrophages play an important role in the innate immune response by phagocytosing IgG-opsonized infectious particles (1) and are major mediators in the destruction of tumor cells (2-5). Indeed, the importance of monocytes in clearing antibody-targeted tumor cells following the administration of therapeutic mAbs used in oncology indications has been well established (6 -8). However, despite showing statistically significant effects, the low rates of complete remission combined with the relatively high relapse rate suggest strongly that there is much room for improvement (9 -11). Therapeutic mAbs themselves are being improved, with the goal of increasing affinity toward Fc␥ receptors in some cases (12, 13). Along with this, immune modulators such as interferons (14, 15), interleukins (16 -20), synthetic compounds (21-23), and CpG oligonucleotides (16) are being explored as potential enhancers of antibody therapy.Antibody-dependent destruction of target cells is largely mediated by Fc␥ receptors (Fc␥Rs) 4 (5, 24, 25). Human monocytes and macrophages express at least four different functional Fc␥Rs: Fc␥RI, Fc␥RIIa, Fc␥RIIb, and Fc␥RIIIa (26). Of these, Fc␥RI, Fc␥RIIa, and Fc␥RIIIa are activating receptors that drive cellular responses to antibodies. These receptors either contain, within their cytoplasmic tails, an immune receptor tyrosine-based activation motif (ITAM), as in the case of Fc␥RIIa (27), or are associated with the ␥-chain homodimer that has an ITAM (28). The association of the ␥-chain is critical not only for surfa...

The Membrane-Associated MARCH E3 Ligase Family: Emerging Roles in Immune Regulation

“…These studies suggest that MARCH2 plays unique inhibitory roles in HIV-1 infection. The transcription of MARCH3 gene is dramatically induced by LPS and TLR8 agonist in monocytes (56). Notably, it has been demonstrated that MARCH1 is highly expressed in human hepatocellular carcinoma (HCC) cells (55), MARCH5 is up-regulated in ovarian cancer tissues (28), and MARCH8 is highly expressed in esophageal tumors and associated with tumor aggression (50).…”

A serine in the first transmembrane domain of the human E3 ubiquitin ligase MARCH9 is critical for down-regulation of its protein substrates

“…MARCH1 has also recently been shown to regulate insulin signaling through control of basal-state insulin receptor levels in adipocytes (26). MARCH2 and MARCH3 are less well-studied, but have been shown to mediate ubiquitin-dependent down-regulation of multispanning membrane proteins such as the ␤ 2 -adrenergic receptor (27) and the cystic fibrosis transmembrane conductance regulator (MARCH2) (28), as well as the inhibitory antibody Fc receptor Fc␥RIIb (MARCH3) (29). The MARCH4 and MARCH9 proteins are closely related to one another and down-regulate MHC-I and the T-cell co-receptor CD4 (1) as well as the natural killer cell activating ligand Mult1 (30).…”

The E3 ubiquitin ligase MARCH3 controls the endothelial barrier

“…Based on the literature, we further focused on MARCH3 (Fig. A, green dots), as it has been previously linked to the endocytosis and trafficking of plasma membrane proteins . The silencing of MARCH3 in human endothelial cells led to a significant reduction in both IL‐8‐ and histamine‐promoted permeability by around 40%, while sparing nonstimulated cells (Fig.…”