A Role for Notch Signaling in the Commitment of Human Blood Monocytes to Langerhans Cells.

We previously demonstrated that Notch ligand Delta-1 in concert with GM-CSF and TGF-β1 promotes the differentiation of human blood monocytes into Langerhans cells that are characterized by the expression of CD1a, Langerhans-associated granules Langerin, cutaneous lymphocyte-associated antigen (CLA), CC chemokine receptor 6 (CCR6), and E-cadherin. These data extended the functional scope of Notch ligand Delta-1 in human adult hematopoiesis. HES-1 is known to be the target gene by Notch signaling. We examined the effect of Delta-1 on the expression of HES-1 mRNA in CD14⁺ blood monocytes in the presence of GM-CSF and TGF-β1, using real-time RT-PCR. When CD14⁺ blood monocytes were cultured with Delta-1, GM-CSF, and TGF-β1, the expression level of HES-1 mRNA increased approximately 10-fold at 24 hours of incubation, compared with the expression level in freshly isolated CD14⁺ monocytes. However, the expression level of HES-1 mRNA declined at 48 hours of incubation. This finding suggests that Delta-1 may operate at the early stage of the differentiation pathway from CD14⁺ monocytes to Langerhans cells. To explore this issue more precisely, we cultured CD14⁺ monocytes in the presence of Delta-1, GM-CSF, and TGF-β1 for 2 days, and subsequently replated the cells into the cultures without Delta-1. At day 7 of culture, cultured cells were harvested and characterized by phenotypic analysis. This initial 2-day exposure of CD14⁺ monocytes to Delta-1 gave rise to Langerhans cells, similar to the observation obtained with the supplementation of Delta-1 throughout 7-day culture. In turn, when Delta-1 was added at day 2 of culture, Langerhans cells were not induced, but instead the resulting cells exhibited the features of macrophages. Our results indicate that in response to Delta-1, human blood monocytes appear to initiate the differentiation program toward Langerhans cells, while they are incapable of differentiating into Langerhans cells after their commitment to macrophages.