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

Abstract 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.

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An Indispensable Role of GM-CSF in the Differentiation of Human Blood Monocytes toward Langerhans Cells.

Blood ◽

10.1182/blood.v106.11.3868.3868 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3868-3868

Author(s):

Tetsunori Shibasaki ◽

Naoyuki Katayama ◽

Kohshi Ohishi ◽

Fumihiko Monma ◽

Zhaocai Yu ◽

...

Keyword(s):

Human Blood ◽

Langerhans Cells ◽

Cultured Cells ◽

Expression Profiles ◽

Blood Monocytes ◽

Similar Action ◽

Gm Csf ◽

Tgf Β1 ◽

Cells Cultured ◽

E Cadherin

Abstract We recently demonstrated that GM-CSF, TGFβ1, and Notch ligand Delta-1, all of which are synthesized in the milieu of the skin, instruct human blood monocytes to differentiate into Langerhans cells (LCs) that are characterized by the expression of CD1a, Langerin, CLA, CCR6, and E-cadherin and the presence of Birbeck granules. Previous studies have shown similar biologic activities of GM-CSF and IL-3 on human blood monocytes. In the present study, we investigated whether GM-CSF and IL-3 have a similar action on blood monocytes with respect to their differentiation into LCs. In contrast to CD14+ monocytes cultured for 7 days in the presence of GM-CSF, TGF-β1, and Delta-1, the cells obtained from culture with IL-3, TGF-β1, and Delta-1 were negative for CLA, CCR6, Langerin, and E-cadherin. These cells expressed HLA-ABC, HLA-DR, CD80, CD86, CD40, CD54, and CD11c but not DC-SIGN. The expression level of CD1a declined, while CD14 was upregulated. The difference between cells cultured with GM-CSF, TGF-β1, and Delta-1 and those with IL-3, TGF-β1, and Delta-1 was corroborated by microarray analysis of the gene expression profiles. Thus, GM-CSF and IL-3 exert distinct effects on human blood monocytes in the presence of TGF-β1 and Delta-1. When GM-CSF was added to cultures containing IL-3, TGF-β1, and Delta-1, the phenotype of cultured cells closely resembled that observed with GM-CSF, TGF-β1, and Delta-1. As GM-CSF and IL-3 share common receptor β subunits, the signals mediated through GM-CSF receptor α subunits appear to be required for the development of LCs from monocytes. We also compared the action of GM-CSF with that of IL-3 in terms of the differentiation of monocytes into macrophages and dendritic cells (DCs). When CD14+ monocytes were cultured in the presence of GM-CSF or IL-3 for 7 days, the resulting macrophages obtained from both cultures were phenotypically indistinguishable. Next, we examined whether IL-3 as well as GM-CSF induces CD14+ monocytes to differentiate into DCs in the presence of IL-4. The phenotypic profiles in cells cultured with IL-3 plus IL-4 were parallel to those seen with DCs cultured in the presence of GM-CSF plus IL-4. These data suggest that although IL-3 can substitute for GM-CSF in the differentiation pathway of CD14+ monocytes toward macrophages or DCs, GM-CSF is indispensable for their LC development.

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The Notch ligand, Delta-1, inhibits the differentiation of monocytes into macrophages but permits their differentiation into dendritic cells

Blood ◽

10.1182/blood.v98.5.1402 ◽

2001 ◽

Vol 98 (5) ◽

pp. 1402-1407 ◽

Cited By ~ 91

Author(s):

Kohshi Ohishi ◽

Barbara Varnum-Finney ◽

Rita E. Serda ◽

Claudio Anasetti ◽

Irwin D. Bernstein

Keyword(s):

Dendritic Cells ◽

Notch Signaling ◽

Cell Fate ◽

Interleukin 4 ◽

Cellular Interactions ◽

Blood Monocytes ◽

Notch Ligand ◽

Cell Fate Decisions ◽

Gm Csf ◽

Tnf Α

Notch-mediated cellular interactions are known to regulate cell fate decisions in various developmental systems. A previous report indicated that monocytes express relatively high amounts of Notch-1 and Notch-2 and that the immobilized extracellular domain of the Notch ligand, Delta-1 (Deltaext-myc), induces apoptosis in peripheral blood monocytes cultured with macrophage colony-stimulating factor (M-CSF), but not granulocyte-macrophage CSF (GM-CSF). The present study determined the effect of Notch signaling on monocyte differentiation into macrophages and dendritic cells. Results showed that immobilized Deltaext-myc inhibited differentiation of monocytes into mature macrophages (CD1a+/−CD14+/− CD64+) with GM-CSF. However, Deltaext-myc permitted differentiation into immature dendritic cells (CD1a+CD14−CD64−) with GM-CSF and interleukin 4 (IL-4), and further differentiation into mature dendritic cells (CD1a+CD83+) with GM-CSF, IL-4, and tumor necrosis factor-α (TNF-α). Notch signaling affected the differentiation of CD1a−CD14+macrophage/dendritic cell precursors derived in vitro from CD34+ cells. With GM-CSF and TNF-α, exposure to Deltaext-myc increased the proportion of precursors that differentiated into CD1a+CD14− dendritic cells (51% in the presence of Deltaext-myc versus 10% in control cultures), whereas a decreased proportion differentiated into CD1a−CD14+ macrophages (6% versus 65%). These data indicate a role for Notch signaling in regulating cell fate decisions by bipotent macrophage/dendritic precursors.

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In-VitroDifferentiation of Mature Dendritic Cells From Human Blood Monocytes

Developmental Immunology ◽

10.1155/1998/72054 ◽

1998 ◽

Vol 6 (1-2) ◽

pp. 25-39 ◽

Cited By ~ 19

Author(s):

Robert Gieseler ◽

Dirk Heise ◽

Afsaneh Soruri ◽

Peter Schwartz ◽

J. Hinrich Peters

Keyword(s):

Dendritic Cells ◽

Human Blood ◽

T Cell Response ◽

Blood Monocytes ◽

Gm Csf ◽

Hla Dr ◽

Hla Dq ◽

Specific Stimulation ◽

Antigen Presenting

Representing the most potent antigen-presenting cells, dendritic cells (DC) can now be generated from human blood monocytes. We recently presented a novel protocol employing GM-CSF, IL-4, and IFN-γto differentiate monocyte-derived DCin vitro. Here, such cells are characterized in detail. Cells in culture exhibited both dendritic and veiled morphologies, the former being adherent and the latter suspended. Phenotypically, they were CD1a-/dim, CD11a+, CD11b++, CD11c+, CD14dim/-, CD16a-/dim, CD18+, CD32dim/-, CD33+, CD40+, CD45R0+, CD50+, CD54+, CD64-/dim, CD68+, CD71+, CD80dim, CD86+/++, MHC class I++/+++HLA-DR++/+++HLA-DP+, and HLA-DQ+. The DC stimulated a strong allogeneic T-cell response, and further evidence for their autologous antigen-specific stimulation is discussed. Although resembling a mature CD 11c+CD45R0+blood DC subset identified earlier, their differentiation in the presence of the Thl and Th2 cytokines IFN-γand IL-4 indicates that these DC may conform to mature mucosal DC.

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Synergistic Interaction between Notch ligand Delta-1 and IL-6/Soluble IL-6 Receptor Fusion Protein in the Generation of BFU-E from Primitive Hematopoietic Progenitors.

Blood ◽

10.1182/blood.v104.11.1626.1626 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1626-1626

Author(s):

Kentaro Yamamura ◽

Kohshi Ohishi ◽

Naoyuki Katayama ◽

Hiroshi Shiku ◽

Masahiro Masuya ◽

...

Keyword(s):

Fusion Protein ◽

Cultured Cells ◽

Hematopoietic Progenitors ◽

Erythroid Progenitors ◽

Isolated Cells ◽

Hematopoietic Stem ◽

Semisolid Medium ◽

Notch Ligand ◽

Gm Csf ◽

Synergistic Enhancement

Abstract We have previously reported that Notch ligand Delta-1 may enhance the generation of NOD/SCID-repopulating hematopoietic stem cells and thymus-repopulating T cell precursors from cord blood [CB] CD34+CD38- cells. A complex of IL-6/soluble IL-6 receptor is shown to induce the expansion and proliferation of primitive hematopoietic progenitors that express gp130 but not the IL-6 receptor α chain, in mobilized peripheral blood (MPB) as well as in CB. In this study, we explored the interaction between Delta-1 and IL-6/soluble IL-6 receptor fusion protein [FP] in the generation of hematopoietic progenitors from MPB- and CB-derived primitive hematopoietic progenitors. MPB CD34+ Thy-1+ cells were cultured in serum-free medium with SCF, flt-3 ligand, TPO, and IL-3 [4GF], 4GF plus Delta-1, 4GF plus FP, and 4GF plus FP plus Delta-1. Delta-1 was used after immobilized to the culture plates. Freshly isolated cells and cultured cells were assessed for colony-forming ability by replating cells into semisolid medium containing SCF, flt-3 ligand, TPO, IL-3, IL-6, G-CSF, GM-CSF, and EPO. Colonies were counted at day 14. The numbers of BFU-E, CFU-Mix, and CFU-GM were 37-fold, 18-fold, and 135-fold increased, respectively, in cultures with 4GF plus FP, as compared with those in freshly isolated cells. The addition of both Delta-1 and FP to cultures resulted in 216-fold, 22-fold, and 132-fold increases in the numbers of BFU-E, CFU-Mix, and CFU-GM, respectively, relative to freshly isolated cells. Delta-1 did not affect the generation of colony-forming cells in cultures without FP. Thus, Delta-1 and FP synergistically enhanced the generation of BFU-E from primitive hematopoietic progenitors in MPB. In the cultures of CB CD34+CD38- cells with SCF, flt-3 ligand, TPO [3GF], 3GF plus Delta-1, 3GF plus FP, and 3GF plus FP plus Delta-1, the synergistic enhancement by Delta-1 and FP was observed in the generation of BFU-E and CFU-Mix. These data suggest a novel role for interaction between Notch and gp130 signalings in the generation of erythroid progenitors from primitive hematopoietic progenitors.

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