IL3 Induces Osteoclastogenesis In Vivo and Is Modulated By Bone Marrow Monocyte / Macrophage Derived Activin A

Abstract

Multiple myeloma (MM) bone disease is characterized by bone destruction with suppressed bone formation resulting in enhanced tumor growth. We previously showed that IL3 is a multifunctional cytokine that is elevated in the marrow plasma of MM patients as compared with healthy subjects and that IL3 increases myeloma cell and osteoclast (OCL) precursor proliferation and suppresses osteoblastogenesis via an indirect mechanism mediated by bone marrow monocyte / macrophages (BMM). In addition, IL3 has widespread proliferative and differentiation effects on multiple hematopoietic cell types. Thus attempts at targeting IL3 to date have been unsuccessful due to IL3's impact on normal hematopoiesis. Therefore, identification of IL3's downstream effects in MM bone disease is critical for effective targeting of IL3 in MM.We demonstrated that IL3 induces human OCL formation and that an IL3 neutralizing antibody inhibits OCL formation stimulated by bone marrow (BM) plasma containing high levels of IL3 from MM patients. We recently reported that treatment of MM patient CD14+ BMM with IL3 induces high levels of Actvin A (ActA), a TGF-β superfamily member that, like IL3, modulates MM bone disease by enhancing osteoclastogenesis and inhibiting osteoblasts. As IL3 induces ActA secretion from CD14+ BMM, and has a very similar effect to IL3 on OCL formation, we treated OCL precursors with IL3 in the presence of a neutralizing antibody to ActA (anti-ActA) and demonstrated that anti-ActA dose-dependently inhibited the osteoclastogenic effect of IL-3 on OCL formation. We also showed that ActA stimulates osteoclastogenesis via a RANKL-independent mechanism, and that, in support of early OCL precursors as the source of ActA induced by IL3, IL3 receptor expression is highest on early OCL precursors and decreases during OCL differentiation. We now report in vivoconfirmation of IL3's osteoclastogenic effects.Female C57BL mice were injected intraperitoneally with saline or anti-ActA for 7 days. Beginning on day 3, mice were injected subcutaneously over the calvaria with IL3 or saline daily for 5 days. Mice were sacrificed after 7 days and calvaria were sectioned and stained for TRAP. A blinded observer performed quantitative histologic analyses, and TRAP+ OCL were counted on the endosteal bone surfaces. TRAP+ OCL numbers on the endosteal bone surfaces (N.Oc), corrected for bone surface area (BS), were dramatically increased by IL3 treatment compared to saline treated controls (mean N.Oc/BS supracalvarial IL3 treatment 18.51, SD 2.14 vs saline control 13.42, SD 1.18, p<0.05.) Further, treatment of mice with anti-ActA prior to and during supracalvarial IL3 treatment significantly reduced N.Oc/BS induced by IL3 (mean N.Oc/BS supracalvarial IL-3 with intraperitoneal anti-ActA 11.01, SD 1.2, p<0.01). These findings are the first demonstration that IL3 induces osteoclastogenesis in vivo and that IL3 induced osteoclastogenesis is completely blocked in vivo by a neutralizing antibody to ActA. These results suggest that therapies targeting ActA activity should block IL3's effects in MM bone disease, and supports investigation of IL3 receptor blockade for the modulation of IL3's downstream effects in MM bone disease. Disclosures:Roodman:Amgen: Consultancy; Eli Lilly and Co.: Research Funding. Giuliani:Celgene Italy: Research Funding.