Neutralization of stem cell factor in vitro and in vivo: dose-dependent inhibition of stromal cells and induction of granulocyte/monocyte differentiation

Abstract

 Stem cell factor (SCF), also termed mast cell growth factor or c-kit ligand, plays a central role in the regulation of hematopoiesis and maintenance of viability of hematopoietic cells. We used a new murine monoclonal antibody (MAb) specific for canine SCF to further dissect the role of SCF in vitro and in vivo. This neutralizing MAb, RG7.6 (IgG1), recognizes the soluble form as well as the membrane-bound form of SCF on marrow-derived stromal cells. Treatment of long-term bone marrow cultures (LTMC) with RG7.6 suppressed or stimulated the production of CFU-GM, depending on the MAb concentration and the time of addition to cultures. At concentrations of 0.1–10 μg/ml given on the day of recharge of the LTMC, RG7.6 resulted in sustained suppression of CFU-GM grown from nonadherent cells. In contrast, higher doses of RG7.6 (20–100 μg/ml) led to a two- to threefold increase in CFU-GM formation from nonadherent cells after 3 days of RG7.6 exposure; after longer RG7.6 exposure there was a rapid decline in the number of CFU-GM. The early increase of CFU-GM was even more distinct when RG7.6 addition to LTMCs was delayed until 1 day before cells were plated for the CFU-GM assay. The early increase of CFU-GMs in the presence of high-dose RG7.6 was mimicked by the addition of granulocyte colony-stimulating factor (G-CSF) to cultures containing suboptimal concentrations of RG7.6, suggesting the possibility that the "positive" response to high-dose RG7.6 was due to an overriding effect of other growth factors, e.g., G-CSF. In stromal cells expressing the membrane-bound form of SCF, the presence of MAb RG7.6, even at low concentrations, interfered with thymidine uptake and proliferation. RG7.6 was also tested in vivo. RG7.6 was given intravenously immediately (days 0–4) after total body irradiation and autologous bone marrow transplantation, and granulocyte counts were followed. The post-irradiation nadir of peripheral blood granulocytes was indistinguishable from controls at low doses of RG7.6 but became more shallow as higher doses of RG7.6 were infused, again suggesting a positive effect on granulocyte differentiation. Thus, the SCF-specific MAb appears to interfere with both stromal and hematopoietic cell function. While only inhibition was observed at lower concentrations, a transient increase in granulocyte production was seen at higher MAb concentrations.