IL-3 Inhibits Osteoblast Formation in Multiple Myeloma Via CD45+ Cells.

Abstract

Normally, osteoclast activation is coupled to an increase in osteoblast activity. In Multiple Myeloma (MM), bone remodeling is uncoupled, and bone destruction occurs both by markedly increased osteoclastic bone destruction and severely impaired osteoblastic bone formation. Although several reports have shown that conditioned media from MM cell lines suppress osteoblast (OBL) differentiation, the identity of the OBL inhibitor(s) is unknown. A recent report by Tian et al (NEJM 2003) has identified DKK1, an inhibitor of the WNT signaling pathway, as a putative OBL inhibitor in MM. However, it is likely that other inhibitors of OBL differentiation are present in the myeloma microenvironment, just as there are several potent stimulators of osteoclast formation produced or induced by myeloma cells. We recently reported that IL-3 levels in bone marrow plasma of patients with MM are increased compared to normal controls and that IL-3 in MM marrow plasma stimulates osteoclast formation. We also demonstrated that IL-3 is produced by primary myeloma cells and that it increased MM cell growth in vitro. However, the effects of IL-3 on OBL are unknown. Therefore, to determine if IL-3 could affect OBL growth and differentiation, we tested the effects of IL-3 on OBL differentiation in primary mouse marrow stromal cells. We found that murine marrow stromal cells and OBL-like cell lines expressed IL-3 receptor alpha (IL-3R) by RT-PCR. Staining for IL-3R in marrow stromal cell cultures after treatment with IL-3 confirmed that the IL-3R was present on thirty percent of the cells. Importantly, treatment of primary murine stromal cell cultures with IL-3 (0.01–10 ng/mL) inhibited basal and BMP-2 stimulated osteoblast formation in a dose dependent manner, without affecting cell growth. At 10 ng/mL IL-3 inhibited OBL differentiation by 80%. Time course studies demonstrated that IL-3 affected the later stages of osteoblast differentiation. Further, the inhibitory effects of IL-3 were not due to induction of TNFalpha, a known OBL inhibitor. TNFalpha levels were very low (0–20pg/ml) in the conditioned media of these cultures, and treating the cultures with anti-mouse TNFalpha did not block the IL-3 effect. IL-3 did not inhibit alkaline phosphatase activity in the osteoblast-like cell lines, MC3T3-E1 and C2C12 suggesting that IL-3 may act indirectly through another cell type in the mixed cell population in the primary mouse osteoblast culture system. Since IL-3 acts primarily on hematopoietic cells, we determined if IL-3 was acting indirectly by increasing CD45+ cells in the cultures. IL-3 increased the number of CD45+ hematopoietic cells in the primary culture from approximately 15% to 30%. Further, depletion of the CD45+ cells abolished the inhibitory effects of IL-3 but not TNFalpha on osteoblasts, and adding back CD45+ cells to the stromal cell cultures restores the inhibitory effects of IL-3. This data suggests that IL-3 is an important mediator of bone destruction in MM by both inducing osteoclast formation and indirectly inhibiting osteoblast formation.