Crosslinking of Fc Gamma-Rllb and Fc Epsilon-RI Binding Peptides Inhibits Osteoclast Formation in Multiple Myeloma through Inactivation of the ITAM Signaling Pathway

Introduction: Overactivity of osteoclasts resulting in bone destruction is a hallmark of multiple myeloma (MM). Receptor for activation of NF-kB ligand (RANKL) and monocyte colony stimulating factor (MCSF) signaling pathways both promote proliferation and survival of the precursors of the osteoclast lineage, and have been widely investigated in MM. The third pathway involved in osteoclast differentiation is the immunoreceptor tyrosine-based activation motif (ITAM) with c-Fms signaling. ITAM and its inhibitor ITIM provide the basis for two opposed signaling modules that duel for control of osteoclast formation. Human monocyte/macrophage expresses the low-affinity FcγRIIb and high-affinity Fcε receptor 1 (FcεRI). Both receptors mediate Syk phosphorylation to activate or inactivate downstream ITAM or ITIM signaling molecules.  In this study, we determined the effects of an IgG_(CH2-CH3) and IgE_{(CH2-CH3-CH4)} fusion protein that activates the ITIM inhibitory pathway on downstream signaling of Syk and osteoclast formation in monocytes from MM patients.

Methods: We constructed IgG_(CH2-CH3) with an IgE_{(CH2-CH3-CH4)} fusion protein using standard cloning techniques.  We evaluated the fusion protein on osteoclast formation using cells from either human monocytes isolated from MM patients' peripheral blood mononuclear cells (PBMCs) or bone marrow (BM) MCs with an anti‑CD14 micro-bead affinity column and magnetic bead selection (Miltenyi Biotec, Auburn, CA). The monocytes were cultured on slide‑culture dishes (2 X 10⁵cells/well). The cells were treated with the fusion protein or with IgE or IgG and subsequently treated with 50ng/ml RANKL (receptor for activation of nuclear factor kB and 10ng/ml MCSF (monocyte colony stimulating factor) in order to stimulate osteoclast formation at the beginning of the culture and during a medium change after 3 days with the same amount of growth factors added. The cells were fixed for tartrate resistant acid phosphatase (TRAP)-staining assay on day 21. To investigate ITIM signaling pathway we determined Syk phosphorylation of monocytes treated or without treated with fusion protein by Western blot analysis.

Results: We found that in a concentration-dependent fashion, the fusion protein inhibited osteoclast cell formation from CD14+ MCs from PB or BM exposed to RANKL and MCSF.  We further analyzed the effects on the FcγRIIb‑SHIP signaling pathway in monocytes induced with 50ng/ml RANKL and 10ng/ml MCSF following exposure to fusion protein or control IgG or IgE. The results showed that the monocytes showed markedly lower Syk phosphorylation following exposure to the fusion protein (100-200ng/ml). There was no change of Syk phosphorylationl in monocytes treated with IgG or IgE or IgG with IgE.

Conclusions: The results of our study show that intact human IgG or IgE does not affect the ITAM or ITIM signaling pathways. However, a fusion protein consisting of IgG_(CH2-CH3) with IgE_{(CH2-CH3-CH4)} showed the ability to activate  the ITIM inhibition pathway through FcγRIIb to reduce osteoclast formation. Thus, blockage of ITAM may be treating novel treatment for preventing bone loss for MM patients.