Evidence that ceramide mediates the ability of tumor necrosis factor to modulate primitive human hematopoietic cell fates

In this study, it is shown that short-term exposure of normal human marrow CD34⁺CD38^- cells to low concentrations of tumor necrosis factor (TNF) in the presence of 100 ng/mL Flt3 ligand and Steel factor and 20 ng/mL interleukin-3 (IL-3), IL-6, and granulocyte colony-stimulating factor, in either bulk or single-cell serum-free cultures, markedly reduces their ability subsequently to generate colony-forming cells (CFCs) in 6-week stromal cell-containing long-term cultures without affecting their viability, mitogenic response, or short-term ability to produce CFCs. A similar differential effect on the functional attributes of CD34⁺CD38^- cells was seen when C2- or C6-ceramide, but not dihydro-C2-ceramide (an inactive analog of ceramide), was substituted for TNF. The addition of D-erythro-MAPP (a specific inhibitor of intracellular ceramide degradation) enhanced the ability of TNF to selectively eliminate long-term culture-initiating cell (LTC-IC) activity. These findings indicate that TNF can directly modulate the ability of CD34⁺CD38^- cells to maintain their LTC-IC function at doses below those required to initiate apoptosis, cell cycle arrest, or both, and they suggest that this may be mediated by the TNF-induced generation of intracellular ceramide. Identification of a signaling intermediate that can influence primitive hematopoietic cell fate decisions offers a new approach to the investigation of signaling mechanisms in normal stem cell populations and to how these may be altered in leukemic cells.