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

Monocytes and macrophages are critical for the effectiveness of monoclonal antibody therapy. Responses to antibody-coated tumor cells are largely mediated by Fc receptors (FcRs), which become activated upon binding to immune complexes. FcRIIb is an inhibitory FcR 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 FcRIIb. Upon further examination, we found that treatment of monocytes with TLR4 agonists could lead to the ubiquitination of FcRIIb protein. A search of our earlier microarray database of monocytes activated with the TLR7/8 agonist R-848 (in which FcRIIb 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 FcRIIb down-regulation. The results showed that LPS activation of TLR4 significantly increased MARCH3 expression and that siRNA against MARCH3 prevented the decrease in FcRIIb following LPS treatment. These data suggest that activation of TLR4 on monocytes can induce a rapid down-regulation of FcRIIb protein and that this involves ubiquitination.