A Novel Switch Culture System for Proliferation and Differentiation of Monocytic Cells: ERK Activation Is Not Sufficient for Macrophage Differentiation by M-CSF.

M-CSF, a ligand for Fms, is an essential cytokine for proliferation and differentiation of monocytes/macrophages. However, molecular events responsible for M-CSF/Fms-mediated differentiation and their differences to proliferation-inducing signals are poorly understood. To clarify the difference, it is required to establish cell culture systems, in which distinct responses (proliferation versus differentiation) can be assessed independently in same cells. Here, we report the establishment of a novel culture system useful for analyzing the differences. TF-1-fms leukemia cells proliferate in response to M-CSF, but tend to differentiate into macrophages in the presence of a phorbol ester TPA. Interestingly, in the presence of TPA, M-CSF was likely to hardly yield the initiation of proliferation signal but markedly accelerate the TPA-triggered macrophage differentiation process. The combination of both TPA and M-CSF caused more drastic morphologic change than the treatment with TPA alone. The cells cultured with TPA and M-CSF showed increased granularity and higher phagocytic activity than those cultured with TPA alone. However, there was no significant difference in the numbers of viable cells and in the percentage of PI-positive dead cells between the two treatments, suggesting that the differentiation-accelerating effect did not reflect the survival-enhancing activity of M-CSF but the factor actively transmitted differentiation signals to TF-1-fms in the presence of TPA. The differentiation by TPA/M-CSF apparently correlated with elevated phosphorylation of ERK, a possible regulator of differentiation in a number of cell systems. However, and of importance, a specific MEK inhibitor, PD98059, inhibited the ERK phosphorylation but not macrophage differentiation. The result raised the possibility that elevated ERK activation was not necessarily sufficient for macrophage differentiation of TF-1-fms cells mediated by M-CSF. The experiments with TF-1-fms cells expressing HIV-1 Nef protein further supported the hypothesis: TF-1-fms cells over-expressing Nef showed impaired macrophage differentiation response but increased ERK phosphorylation (Suzu S et al., Blood 2005). Therefore, our switch culture system provides a useful tool for identifying molecules regulating the M-CSF/Fms-mediated macrophage differentiation.