Biological characterization of a partially purified negative regulator of hematopoiesis, NRH

We have identified and partially characterized a negative regulator of human hematopoiesis with novel properties secreted from a new human macrophage line, 2MAC (Blood 86,69la, 1995). The 2MAC conditioned medium is inhibitory for BFUE, CFU-GM, ai well as CFU-GEMM colonies in vitro. In contrast, colony formation of CFU-E and normal T cells is little affected. Using highly purified human CD34 cells, it was shown that this negative regulatory activity suppresses in vitro growth of HPP-CFC in a dose-dependent manner. We demonstrate similar inhibitory activities in material partially purified by different purification schemes, indicating that the negative activity present in the conditioned medium is not likely due to synergistic suppression arising from a combination of more than one negative regulator. The inhibitory activity was shown to be reversible and to correspond to a 20kDa heparin-binding, GlcNAccontaining glycoprotein. This macrophage-derived inhibitory factor can be distinguished from TGF-, MIP-lct and other known inhibitors by a number of criteria including the lack of neutralization by respective specific antibodies. It was also shown that the mechanism of action for the 2MAC-secreted inhibitory activity in either conditioned medium or partially purified material involves a marked decrease in the cycling of hcmatopoictic stem/early progenitor ceils. The percentage of HPP-CFCs from CD34+ cells in S phase of cell cycle decreases from 33.3 ±2.7 % to 5.3 ±0.4 % and the percentage of DNA-synthcsizing BFU-Es is also dropped from 46.5 ±2.1 % to 6.3 ± 0.3 % (a total of 4 determinations) on incubation with partially purified NRH recovered from a Q-Sepharose column. Addition of this partially purified material also abrogates the ability of fresh medium to stimulate cell cycling of the primitive early progenitors in IhcLTC adherent layer (47.1 ±2.0%vs 12.8± i .0 % in 4 determinations).