Journal of Leukocyte Biology BioLegend: Treg, Th17, Stem Cell
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.1204746 on July 21, 2005

Published online before print July 21, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.1204746v1
78/4/921    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hoshino, N.
Right arrow Articles by Shiku, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hoshino, N.
Right arrow Articles by Shiku, H.
(Journal of Leukocyte Biology. 2005;78:921-929.)
© 2005 by Society for Leukocyte Biology

A novel role for Notch ligand Delta-1 as a regulator of human Langerhans cell development from blood monocytes

Natsuki Hoshino*, Naoyuki Katayama*,1, Tetsunori Shibasaki*, Kohshi Ohishi*, Junji Nishioka{dagger}, Masahiro Masuya{ddagger}, Yoshihiro Miyahara*, Masahiko Hayashida§, Daiki Shimomura§, Takuma Kato, Kaname Nakatani{ddagger}, Kazuhiro Nishii*, Kagemasa Kuribayashi, Tsutomu Nobori{dagger} and Hiroshi Shiku*

* Departments of Hematology and Oncology,
{dagger} Laboratory Medicine, and
Bioregulation, Mie University School of Medicine, Tsu, Japan;
{ddagger} Blood Transfusion Service, Mie University Hospital, Tsu, Japan; and
§ Tenri Institute of Medical Research, Tenri, Japan

1Correspondence: Department of Hematology and Oncology, Mie University School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan. E-mail: n-kata{at}clin.medic.mie-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human Langerhans cells (LCs) are of hematopoietic origin, but cytokine regulation of their development is not fully understood. Notch ligand Delta-1 is expressed in a proportion of the skin. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor-ß1 (TGF-ß1) are also secreted in the skin. We report here that Delta-1, in concert with GM-CSF and TGF-ß1, induces the differentiation of human CD14+ blood monocytes into cells that express LC markers: CD1a, Langerin, cutaneous lymphocyte-associated antigen, CC chemokine receptor 6, E-cadherin, and Birbeck granules. The resulting cells display phagocytic activity and chemotaxis to macrophage inflammatory protein-1{alpha} (MIP-1{alpha}). In response to CD40 ligand and tumor necrosis factor {alpha}, the cells acquire a mature phenotype of dendritic cells that is characterized by up-regulation of human leukocyte antigen (HLA)-ABC, HLA-DR, CD80, CD86, CD40, and CD54 and appearance of CD83. These cells in turn show chemotaxis toward MIP-1ß and elicit activation of CD8+ T cells and T helper cell type 1 polarization of CD4+ T cells. Thus, blood monocytes can give rise to LCs upon exposure to the skin cytokine environment consisting of Delta-1, GM-CSF, and TGF-ß1, which may be, in part, relevant to the development of human epidermal LCs. Our results extend the functional scope of Notch ligand {delta}-1 in human hematopoiesis.

Key Words: granulocyte-macrophage colony-stimulating factor· • transforming growth factor ß1 • CD14


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Langerhans cells (LCs), one of the cell populations within the epidermis, contribute to the initiation of primary T cell immune responses [1 2 3 4 5 ]. LCs take up and process antigens that enter through the skin and migrate from the epidermis into the dermis. They subsequently home to the regional lymph nodes via lymphatic vessels. During this process, LCs undergo maturation and acquire the potential to activate antigen-specific naïve T cells. Clinical investigations [6 , 7 ] and in vitro studies [8 9 10 ] indicated that human postnatal LCs arise from hematopoietic progenitor cells in the bone marrow. A number of intermediate precursor cells between hematopoietic progenitor cells and epidermal-resident LCs have been identified in human adults: CD14+ blood monocytes [11 12 13 ], CD1a+CD11c+ blood dendritic cells (DCs) [14 ], and CD14+ cells in the dermis [15 ].

Notch signaling, which is initiated through receptor-ligand interactions, plays a crucial role in the cell-fate decisions and differentiation processes in various organisms [16 17 18 19 ]. Prior studies have identified four receptors, Notch-1 through -4, and five ligands, Jagged-1 and -2 and Delta-1, -3, and –4, in mammals. In the human hematopoietic system, activation of Notch signaling is implicated in proliferation and differentiation of hematopoietic cells. Jagged-1 and Delta-1 induce the expansion of human primitive hematopoietic progenitor cells [20 , 21 ]. Jagged-1 also inhibits the growth of human myeloid progenitors in vitro [22 , 23 ]. The Notch/Notch ligand system seems to be active on monocytes. In fact, the Notch signaling inhibits survival of human monocytes and permits their differentiation into dermal-type DCs, depending on the type of cytokines [24 , 25 ].

Keratinocytes in the epidermis secrete transforming growth factor-ß1 (TGF-ß1) [26 , 27 ]. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is produced by a variety of cells that populate the skin [28 , 29 ]. Monocytes, which originate in the bone marrow, enter the peripheral blood and constitute ~5% of circulating white blood cells. In response to appropriate stimuli, they migrate from the bloodstream into various peripheral tissues including the skin. It was reported that Notch ligand Delta-1 is expressed by skin-resident cells [30 ]. Here, we have investigated the effect of Notch ligand Delta-1 on human blood monocytes in the presence of GM-CSF and TGF-ß1, both cytokines synthesized in the milieu of the skin. The results show that Notch ligand Delta-1, in cooperation with GM-CSF and TGF-ß1, promotes the differentiation of monocytes into LCs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell preparation
Peripheral blood was obtained from healthy adult Japanese donors who all provided written, informed consent. Peripheral blood mononuclear cells (PBMCs) were separated by centrifugation on Ficoll-Hypaque, washed with Ca2+- and Mg2+-free phosphate-buffered saline (PBS), and suspended in PBS with 0.1% bovine serum albumin (BSA; Sigma Chemical Co., St. Louis, MO). CD14+ cells were isolated from PBMCs by positive magnetic separation using CD14 immunomagnetic beads and magnetic separation columns [magnetic cell sorter (MACS); Miltenyi Biotec, Auburn, CA], according to the manufacturer’s instructions. The purity of CD14+ cells exceeded 95%, as determined by a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). When stained with myeloperoxidase (MPO) staining kits and double-specific (naphthol AS-D chloroacetate esterase)/nonspecific ({alpha}-naphthyl butyrate esterase) esterase staining kits, the CD14+ cells were positive for MPO and nonspecific esterase but negative for specific esterase. CD8+ T cells were separated from PBMCs using CD8 immunomagnetic beads (MACS, Miltenyi Biotec). Umbilical cord blood samples were obtained according to institutional guidelines. CD4+ T cells were purified from cord blood using CD4 immunomagnetic beads (MACS, Miltenyi Biotec). The purity of CD8+ T or CD4+ T cells routinely exceeded 95%.

Cytokines and {gamma}-secretase inhibitor
The extracellular domain of Delta-1 was immobilized and used as Notch ligand Delta-1, as described [24 , 31 ]. The construct containing cDNAs of the extracellular domain of Delta-1 and six myc epitopes was inserted into NSO myeloma cells. The extracellular domain of Delta-1 with six myc-tags (Delta-1ext-myc) was purified from conditioned medium. Delta-1ext-myc (1 µg/mL) was added to the 24-well tissue-culture plates (Nunc, Roskilde, Denmark), which were incubated with F(ab')2 fragments of a mouse anti-myc antibody, 9E10, at the concentration of 5 µg/mL for 30 min at 37°C. The culture plates were washed with PBS. Delta-1ext-myc and 9E10 were gifts of Dr. Irwin D. Bernstein (Fred Hutchinson Cancer Research Center, Seattle, WA). Recombinant human (rh)GM-CSF was a gift from Kirin Brewery (Tokyo, Japan). rhTGF-ß1, macrophage inflammatory protein-3{alpha} (MIP-3{alpha}), and MIP-3ß were purchased from R&D Systems (Minneapolis, MN). Recombinant interleukin-4 (IL-4) was purchased from Genzyme (Cambridge, MA). rhCD40 ligand (rhCD40L) was purchased from Bender MedSystems (Vienna, Austria). rh tumor necrosis factor {alpha} (TNF-{alpha}) was a gift from Dainippon Pharmaceutical (Suita, Japan). rhIL-2 was a gift from Takeda Pharmaceutical (Osaka, Japan). Cytokines were used at the following concentrations: GM-CSF, 10 ng/mL; TGF-ß1, 10 ng/mL; MIP-3{alpha}, 100 ng/mL; MIP-3ß, 100 ng/mL; IL-4, 10 ng/mL; CD40L, 1 µg/mL; TNF-{alpha}, 20 ng/mL; IL-2, 20 IU/mL. The Notch signals are activated via Notch cleavage, which is blocked by inhibition of {gamma}-secretase activity [32 , 33 ]. To inhibit Notch signals, {gamma}-secretase inhibitor II (Calbiochem, La Jolla, CA) was used. This inhibitor was dissolved initially in dimethyl sulfoxide (DMSO), diluted in culture medium, and added to the cultures at the final concentration of 30 µM. An equal volume of DMSO was added to control cultures.

Culture
Culture medium was RPMI 1640 (Nissui Pharmaceutical, Tokyo, Japan), supplemented with 2 mM L-glutamine, 50 U/mL penicillin, 50 µg/mL streptomycin, and 10% fetal bovine serum (FBS; HyClone Laboratories, Logan, UT). CD14+ cells (5x105/mL) were cultured with designated combinations of cytokines for 7 days. Half of the culture medium was replaced with fresh medium containing cytokines on Day 3 or 4. In some experiments, CD40L and TNF-{alpha} were added to cultures on Day 5 of incubation.

Flow cytometric analysis
Flow cytometric analysis was done using a FACSCalibur flow cytometer. CellQuest software (Becton Dickinson) was used for data acquisition and analysis. Freshly isolated and cultured cells were reacted with monoclonal antibodies (mAb) for 30 min on ice and washed three times with PBS. After the final wash, cells were incubated with 1 µg/mL propidium iodide to gate out dead cells. The following murine mAbs were used for staining of cell-surface molecules: phycoerythrin (PE)-conjugated anti-CD1a (anti-CD1a-PE), anti-Langerin (CD207)-PE, unconjugated anti-E-cadherin, anti-CD40-PE, and anti-CD83-PE (Immunotech, Marseille, France); anti-human leukocyte antigen (HLA)-ABC-PE and fluorescein isothiocyanate (FITC)-conjugated anti-CD1a (anti-CD1a-FITC; Dako, Glostrup, Denmark); anti-HLA-DR-PE, anti-CD80-PE, and anti-CD54-PE (Becton Dickinson); anti-CD86-PE, anti-CD14-PE, anti-CC chemokine receptor 7 (CCR7)-PE, and anti-DC-specific intercellular adhesion molecule-grabbing nonintegrin (SIGN; CD209)-PE (BD PharMingen, San Diego, CA); and anti-CCR6-FITC (R&D Systems). An anti-cutaneous lymphocyte-associated antigen (CLA)-FITC rat mAb (BD PharMingen) was also used. Mouse immunoglobulin G1 (IgG1)-PE, mouse IgG1-FITC, rat anti-mouse IgG1-FITC, and rat IgG2a-PE were purchased from BD Biosciences (San Jose, CA). Purified mouse IgG1 (R&D Systems), rat IgM-FITC, mouse IgG2b-PE (BD PharMingen), mouse IgG2a-PE (Becton Dickinson), or mouse IgG2b-FITC (Beckman Coulter, Hialeah, FL) served as an isotype control. Double-staining was performed with anti-CD1a-FITC and anti-Langerin-PE, anti-CD1a-PE and anti-CCR6-FITC, or anti-CD1a-PE, unconjugated anti-E-cadherin, and rat anti-mouse IgG1-FITC.

Phagocytosis
Cultured cells (2x105/mL) were incubated with FITC-dextran (molecular weight 40,000, Sigma Chemical Co.) for 1 h at 37°C or on ice in 10% FBS RPMI 1640. The reaction was halted by addition of cold 1% FBS PBS. After washing three times with 1% FBS PBS, uptake of FITC-dextran by the cells was analyzed on a FACSCalibur flow cytometer [34 ].

Chemotaxis assay
Chemotaxis of cultured cells was evaluated using a transwell chemotaxis technique (a 5-µm pore, 6.5-mm polycarbonate transwell plate, Corning Inc., Corning, NY) [35 ]. RPMI 1640 with 0.5% BSA was used as an assay medium. A volume of 600 µl 0.1% BSA PBS containing MIP-3{alpha} (100 ng/mL) or MIP-3ß (100 ng/mL) or the same volume of 0.1% BSA PBS was added to the lower compartment. Cultured cells (5x104) in 100 µl were applied to the upper compartment. The plates were incubated at 37°C in a humidified atmosphere with 5% CO2 for 2 h. The number of cells that migrated to the lower compartment was counted using a Neubauer hematocytometer (American Optical, Buffalo, NY). Each assay was conducted in triplicate. The results were expressed as percentage of migrated cells as a proportion of input cells.

Carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling
Freshly isolated CD14+ cells (1x106/mL) were incubated for 10 min at 37°C with 0.5 µM CFSE (Molecular Probes, Eugene, OR), a cytoplasmic dye that is equally diluted between daughter cells. The cells were washed three times with 10% FBS RPMI 1640 and cultured as described above. CFSE-labeled, freshly isolated and cultured cells were analyzed for fluorescence intensity using a FACSCalibur flow cytometer [36 , 37 ].

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR)
Real-time RT-PCR was done as described [21 ]. Total RNA was extracted from freshly isolated CD14+ cells and the CD14+ cells, which were cultured in the presence of Delta-1, GM-CSF, and TGF-ß1 for 24 h, using an RNeasy blood mini kit (Qiagen, Tokyo, Japan). cDNAs were synthesized by Omniscript RT (Qiagen) using oligo dT primers according to the manufacturer’s instruments. Primers and probes were as follows: hairy and enhancer of split (HES)-1 forward primer, 5'-TGG AAA TGA CAG TGA AGC ACC-3', HES-1 reverse primer, 5'-GTT CAT GCA CTC GCT GAA GC-3', HES-1 probe, 5'-(FAM)-CGC AGA TGA CGG CTG CGC TG-[carboxytetramethylrhodamine (TAMRA)]-3'; the endogenous gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), forward primer, 5'-GAA GGT GAA GGT CGG AGT-3', GAPDH reverse primer, 5'-GAA GAT GGT GAT GGG ATT TC-3', GAPDH probe, 5'-(FAM)-TTG CCA TCA ATG ACC CCT TCA TTG AC-(TAMRA)-3'. Amplifications were carried out in an ABI PRIAM 7700 sequence detector (Applied Biosystems, Foster City, CA) for 2 min at 50°C, 10 min at 95°C, followed by 50 cycles of 15 s at 95°C and 1 min at 60°C. The CD14+ cells, which were cultured in the presence of the conditioned media collected from nontransfected NSO cells, served as a control. To determine whether the products were HES-1, the RT-PCR products were sequenced using an automatic DNA sequencer (Applied Biosystems 373; Applied Biosystems), as described previously [38 ].

RT-PCR
The expression of CCR7 in cultured cells was analyzed by RT-PCR, as described [38 , 39 ]. The primers were used as follows: the forward primer, 5'-GATTACATCGGAGACAACACC-3'; the reverse primer, 5'-TAGTCCAGGCAGAAGAGTCG-3'. GAPDH was used as an internal control. PCR was done for 35 cycles under the following conditions: 94°C for 1 min, 61.5°C for 2 min, and 72°C for 3 min. The PCR products were electrophoresed in 3% agarose gel and stained with ethidium bromide for viewing.

Electron microscopy
CD14+ cells were cultured with Delta-1, GM-CSF, and TGF-ß1. The cultured cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) and postfixed with 1% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.4). Next, the cells were dehydrated through a graded series of ethanol and embedded in Epon. Ultrathin sections were stained with lead citrate and uranyl acetate and examined under a JEM-1200EX electron microscope (Japan Electron Optics Laboratory, Tokyo, Japan).

Enzyme-linked immunospot (ELISPOT) assay
The ELISPOT assay was carried out as described previously but with some modifications [40 ]. As a modified Melan-A26–35 peptide (A27L) was reported to show higher HLA-A*0201 binding affinity than did the natural epitope [41 ], we used the modified peptide, ELAGIGILTV, synthesized by Mitsubishi Chemical (Yokohama, Japan). Ninety-six-well ELISPOT plates (Millipore, Bedford, MA) were coated with anti-human interferon-{gamma} (IFN-{gamma}) mAb (1-D1K; Mabtech, Stockholm, Sweden; 15 µg/mL) overnight at 4°C, washed with PBS, and incubated with 10% human AB serum RPMI 1640 for 2 h at 37°C. Mature LCs were generated from CD14+ cells in the presence of Delta-1, GM-CSF, and TGF-ß1, followed by the supplementation of CD40L and TNF-{alpha} for the last 2 days of culture. The mature LCs were pulsed with 10 µM of the modified peptides. CD8+ T cells (2x105/well) were stimulated with autologous, irradiated (46 Gy), peptide-pulsed, mature LCs (4x104/well) in 10% human AB serum RPMI 1640 for 10 days. Half of the culture medium was replaced with 10% human AB serum RPMI 1640 supplemented with 20 IU/mL IL-2 on Days 4 and 7 of incubation. On day 10, the cultured CD8+ T cells were harvested and used as effecter cells. T2 cells, pulsed with the modified peptides, were used as target cells. T2 cells are the TxB hybrid blast line, which is deficient in transporter associated with antigen processing [42 ]. Nonpulsed T2 cells were included as negative controls. Effecter cells (1x104) and target cells (5x104) were seeded on each well of the ELISPOT plate. The assays were done in triplicate. After 18 h incubation at 37°C with 5% CO2, the plates were washed with PBS containing 0.05% Tween 20 (PBS-Tween) and reacted with biotinylated anti-human IFN-{gamma} mAb 7-B6-1 (Mabtech; 0.2 µg/mL) in PBS for 2.5 h at 37°C. After extensive washing with PBS-Tween, the plates were incubated with streptavidin-conjugated alkaline phosphatase for 90 min at room temperature. Each well was washed with PBS-Tween and then stained using an alkaline phosphatase-conjugate substrate kit (BioRad, Hercules, CA) for visualization of spots. The reaction was halted by rinsing the plates with distilled water. The numbers of spots were counted under a dissecting microscope.

Intracellular cytokine production of CD4+ T cells
Mature LCs (2x105) were cocultured with allogeneic CD4+ T cells (2x105) derived from cord blood in the presence of anti-CD3 mAb (UCHT1, 0.5 µg/mL, BD PharMingen) in 24-well tissue-culture plates. The culture medium was replaced with 10% human AB serum RPMI 1640 supplemented with 20 IU/mL IL-2 on Day 4 of incubation. On Day 7, the cells were washed with PBS and stimulated with phorbol 12-myristate 13-acetate (PMA; 50 ng/mL, Sigma Chemical Co.) and ionomycin (1 µM, Sigma Chemical Co.) for 5 h. After incubated with brefeldin A (5 mg/mL, Sigma Chemical Co.) for 3 h to prevent cytokine secretion, the cells were stained with anti-CD4-allophycocyanin (BD PharMingen). After washing, intracellular IFN-{gamma} and IL-4 were stained with anti-IFN-{gamma}-FITC and anti-IL-4-PE (BD PharMingen), respectively, using a cytofix/cytoperm kit (PharMingen). Mouse IgG1-FITC or rat IgG1-PE (BD PharMingen) was used as an isotype control. Flow cytometric analysis was done using a FACSCalibur flow cytometer.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of LCs from CD14+ monocytes in the presence of Delta-1, GM-CSF, and TGF-ß1
An earlier study reported that a small population of CD14+ monocytes cultured with GM-CSF and TGF-ß1 expresses LC-related antigens such as Langerin [13 ]. We cultured CD14+ monocytes in the presence of GM-CSF and TGF-ß1 for 7 days. The expression of LC-related antigens, including CD1a, CLA, CCR6, Langerin, and E-cadherin, was not observed (Fig. 1A ). The cells were positive for HLA-ABC, HLA-DR, CD80, CD86, CD40, CD54, and CD14 but negative for CD83, CCR7, and DC-SIGN. We also cultured CD14+ monocytes in the presence of GM-CSF, IL-4, and TGF-ß1 for 7 days. The cultured cells exhibited the phenotype of CD1a+, CLA+, CCR6–, Langerin–, E-cadherin–, HLA-ABC+, HLA-DR+, CD80+, CD86low, CD40+, CD54+, CD83–, CD14–, CCR7–, and DC-SIGN+ (Fig. 1B) , which suggested that these cells are monocyte-derived DCs with the exception of CCR6 expression, inconsistent with the results observed by other investigators [11 ]. GM-CSF and TGF-ß1 are secreted from the skin-resident cells [26 27 28 29 ]. Recently, it has been shown that Notch ligand Delta-1 is expressed in part of the epidermis [30 ]. We cultured CD14+ monocytes with Delta-1 as well as GM-CSF and TGF-ß1 for 7 days. When morphologic appearance of the cultured cells was examined on an inverted microscope, formation of loosely adherent clusters was observed (Fig. 2A ). A small number of cells were spindle-shaped. On May-Grunwald-Giemsa staining, the cells were characterized by a large-size, oval-shaped nucleus and irregular cytoplasmic membrane (Fig. 2B) . The nucleus had a dispersed chromatin. The cytoplasm was homogenous with a few vacuoles. Phenotypic features of the cells are shown in Figure 2C . The cells expressed a high level of CD1a and moderate levels of CLA and CCR6. Langerin and E-cadherin were detected in a sizeable fraction of the cultured cells. The ranges of Langerin+ and E-cadherin+ cells were 88–93% and 54–72%, respectively. The cells were positive for HLA-ABC, HLA-DR, CD80, CD86, CD40, CD54, and CD14 but negative for CD83, CCR7, and DC-SIGN. To further characterize the resulting CCR6+, Langerin+, or E-cadherin+ cells, the expression of CD1a on these cells was determined by double-staining. The CCR6+, Langerin+, and E-cadherin+ cells exclusively expressed CD1a (Fig. 2D) . By transmission electron microscopy, the cells exhibited long, prominent dendrites homogenously distributed on the cell surface and contained multiple organelles (Fig. 2E) . Rod- or racket-shaped Birbeck granules with central lamella were detected within the cytoplasm, especially at the marginal region and around the Golgi apparatus. These Birbeck granules were encountered in ~30% of the cultured cells when respective sections from >100 cells were analyzed. The cell profiles in which the presence of Birbeck granules was evident contained two to 40 Birbeck granules. Taken together, these data demonstrate that in the presence of Delta-1, GM-CSF, and TGF-ß1, CD14+ monocytes give rise to cells with features of LCs. However, it is possible that these LCs might originate from a rare population of hematopoietic progenitor cells with a high proliferation potential that contaminated the CD14+ monocyte population [8 , 9 ]. To exclude this possibility, we irradiated CD14+ monocytes at 15 Gy before culturing with Delta-1, GM-CSF, and TGF-ß1. The irradiation did not alter the generation of LCs from CD14+ monocytes (data not shown). We confirmed no cell division throughout the culture of CD14+ monocytes in the presence of Delta-1, GM-CSF, and TGF-ß1 using CFSE labeling (data not shown). To examine whether the cells with morphologic and phenotypic features of LCs generated from CD14+ monocytes in cultures with Delta-1, GM-CSF, and TGF-ß1 have the phagocytic function, we studied their uptake of FITC-dextran. The LCs exhibited the phagocytic activity, as analyzed by flow cytometry (Fig. 2F) . We tested chemotaxis of LCs toward MIP-3{alpha} and MIP-3ß using transwell plates. The chemotaxis in response to MIP-3{alpha} but not MIP-3ß was found (Fig. 2G) . Delta-1 was reported to induce apoptosis of CD14+ cells in culture [24 ]. In our culture system, viability of the recovered cells was routinely >90%.



View larger version (29K):
[in this window]
[in a new window]
  		Figure 1. Phenotype of CD14+ monocytes cultured in the presence of GM-CSF and TGF-ß1 (A) or GM-CSF, IL-4, and TGF-ß1 (B). CD14+ monocytes (5x105/mL) were cultured for 7 days. The expression of CD1a, CLA, CCR6, Langerin, E-cadherin HLA-ABC, HLA-DR, CD80, CD86, CD40, CD54, CD83, CD14, CCR7, and DC-SIGN was analyzed using a FACSCalibur flow cytometer. The thick and thin lines represent the expression of the indicated molecules and isotype controls, respectively. Similar results were obtained with three different donors.

 


View larger version (56K):
[in this window]
[in a new window]
  		Figure 2. Generation of LCs from CD14+ monocytes cultured in the presence of Delta-1, GM-CSF, and TGF-ß1. CD14+ monocytes (5x105/mL) were cultured in the presence of Delta-1, GM-CSF, and TGF-ß1 for 7 days. (A) Cultured cells were examined under an inverted microscope (original magnification, x400). (B) May-Grunwald-Giemsa-stained cytospin preparations of the cultured cells were observed on light microscopy (original magnification, x400). (C) The expression of CD1a, CLA, CCR6, Langerin, E-cadherin, HLA-ABC, HLA-DR, CD80, CD86, CD40, CD54, CD83, CD14, CCR7, and DC-SIGN by the cultured cells was analyzed using a FACSCalibur flow cytometer. The thick and thin lines represent the expression of the indicated molecules and isotype controls, respectively. (D) Two-color flow cytometric analysis of the cultured cells was performed using a FACSCalibur flow cytometer: CCR6 and CD1a; Langerin and CD1a; E-cadherin and CD1a. (E) Ultrastructure of the cultured cells was studied at low (left panel: original magnification, x8000; bar, 2 µm) and high (right panel: original magnification, x30,000; bar, 0.2 µm) powers. Arrowheads point to Birbeck granules. (F) Phagocytic activity of LCs was studied using FITC-dextran. LCs generated from CD14+ monocytes in cultures with Delta-1, GM-CSF, and TGF-ß1 were incubated with FITC-dextran for 1 h at 37°C or on ice. The cells were washed with 1% FBS cold PBS and analyzed using a FACSCalibur flow cytometer. (G) Migratory responses to MIP-3{alpha} and MIP-3ß of the cultured cells. Control culture contains the same volume of 0.1% BSA PBS. Numbers of cells that migrated to the lower chamber were counted. The assays were performed in triplicate. Results represent the mean ± SD of percentage of migrated cells to input cells. *, P < 0.001, compared with control. Similar results were obtained with three different donors. Recovery of cultured cells was 31.4% ± 6.2% of input cells (n=5; three different donors).

 
HES-1 gene expression induced by Delta-1
HES-1 is known to be the target gene by Notch signaling [43 , 44 ]. We examined the effect of Delta-1 on the expression of HES-1 mRNA to determine whether the Notch signaling mediated by Delta-1 operated in the generation of LCs in our culture system. We cultured freshly isolated CD14+ monocytes with GM-CSF and TGF-ß1 in the presence or absence of Delta-1 and analyzed the expression levels of HES-1 by the cultured cells and freshly isolated CD14+ monocytes using real-time RT-PCR (Fig. 3 ). Delta-1 induced more than a tenfold increase in HES-1 mRNA expression, compared with the expression level in freshly isolated CD14+ monocytes. The expression level of HES-1 mRNA in cells cultured in the absence of Delta-1 did not alter compared with that of freshly isolated CD14+ monocytes. This increase in HES-1 mRNA expression induced by Delta-1 indicates that Notch signaling mediated by Delta-1 functioned in the generation of LCs in our culture system. Furthermore, we confirmed that the RT-PCR products were HES-1 by using a sequencing technique.



View larger version (10K):
[in this window]
[in a new window]
  		Figure 3. Up-regulation of HES-1 mRNA in CD14+ monocytes by Delta-1. CD14+ monocytes (5x105/mL) were cultured with GM-CSF and TGF-ß1 in the presence of Delta-1 or the control-conditioned medium. After 24 h, the expression level of HES-1 mRNA was determined by real-time RT-PCR, relative to that of GAPDH mRNA used as an internal control. The expression level in freshly isolated CD14+ monocytes was considered as the 1.0 value. Similar results were obtained with two different donors.

 
Effect of a {gamma}-secretase inhibitor on the generation of LCs from CD14+ monocytes in the presence of Delta-1, GM-CSF, and TGF-ß1
The {gamma}-secretase inhibitor II has been reported to inhibit the Notch signal by blocking Notch cleavage [32 , 33 ]. To confirm that the Notch signal induced by Delta-1 is necessary for LC development, we examined the effect of {gamma}-secretase inhibitor II on the generation of LCs from CD14+ monocytes supported by Delta-1, GM-CSF, and TGF-ß1 (Fig. 4 ). The generation of LCs was evaluated by the expression of CD1a, CLA, CCR6, Langerin, E-cadherin, CD14, HLA-ABC, and HLA-DR. The cells cultured in the presence of the {gamma}-secretase inhibitor did not express CD1a, CLA, CCR6, Langerin, or E-cadherin, and the expression of CD14, HLA-ABC, and HLA-DR was retained. These results indicate that Delta-1 is essentially required for LC development from CD14+ monocytes in our culture system.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 4. Inhibition of LC generation from CD14+ monocytes in the presence of Delta-1 by addition of a {gamma}-secretase inhibitor. CD14+ monocytes (5x105/mL) were cultured with Delta-1, GM-CSF, and TGF-ß1 in the presence of {gamma}-secretase inhibitor II (30 µM) or an equal volume of vehicle (DMSO) as a control. After 7 days, the expression of CD1a, CLA, CCR6, Langerin, E-cadherin, CD14, HLA-ABC, and HLA-DR was analyzed using a FACSCalibur flow cytometer. The thick and thin lines represent the expression of the indicated molecules and isotype controls, respectively. Similar results were obtained with three different donors.

 
Phenotypic maturation of LCs generated from CD14+ monocytes in cultures with Delta-1, GM-CSF, and TGF-ß1
We examined whether LCs generated from CD14+ monocytes in our cultures could acquire a mature phenotype in response to CD40L and TNF-{alpha}. We cultured CD14+ monocytes in the presence of Delta-1, GM-CSF, and TGF-ß1 and subsequently added CD40L and TNF-{alpha} to the cultures. The resulting cells showed a significantly increased expression of HLA-ABC, HLA-DR, CD80, CD86, CD40, and CD54 and acquired the expression of CD83, a finding consistent with phenotypic maturation (Fig. 5A ). The expression of Langerin and CD14 was retained. CCR6 expression was down-regulated considerably, and the cells expressed CCR7 at only a marginal level. The expression of DC-SIGN remained undetectable.



View larger version (31K):
[in this window]
[in a new window]
  		Figure 5. Phenotype, chemotaxis, and T cell stimulation of mature LCs. (A) Phenotype of LCs generated from CD14+ monocytes on exposure to CD40L and TNF-{alpha}. CD14+ monocytes (5x105/mL) were cultured in the presence of Delta-1, GM-CSF, and TGF-ß1, followed by the delayed addition of CD40L and TNF-{alpha} on Day 5 of culture. On Day 7 of incubation, the expression of Langerin, CCR6, HLA-ABC, HLA-DR, CD80, CD86, CD40, CD54, CD83, CD14, CCR7, and DC-SIGN was analyzed using a FACSCalibur flow cytometer. The thick and thin lines represent the expression of the indicated molecules and isotype controls, respectively. Similar results were repeated in five experiments (three separate donors). (B) Migratory responses to MIP-3{alpha} and MIP-3ß of the cultured cells. Control culture contains the same volume of 0.1% BSA PBS. Numbers of cells that migrated to the lower chamber were counted. The assays were performed in triplicate. Results represent the mean ± SD of percentage of migrated cells to input cells. *, P < 0.001, compared with control. Similar results were obtained with three different donors. (C) Mature LCs generated from CD14+ monocytes activate peptide-specific CD8+ T cells. CD14+ monocytes were obtained from three different HLA-A*0201+ healthy donors (HD). Mature LCs were generated from CD14+ monocytes in culture with Delta-1, GM-CSF, and TGF-ß1, followed by the delayed addition of CD40L and TNF-{alpha}. Autologous CD8+ T cells (2x105/well) were stimulated with mature-irradiated (46 Gy) LCs (4x104/well), pulsed with the modified peptide Melan-A26–35 (A27L) for 10 days. CD8+ T cells (1x104) and peptide-pulsed T2 cells (5x104) were placed in each well of the ELISPOT plates. After 18 h incubation, the plates were reacted with biotinylated anti-human IFN-{gamma} mAb and incubated with streptavidin-conjugated alkaline phosphatase. Each well was stained with an alkaline phosphatase conjugate substrate kit for visualization of spots. The numbers of spots were counted under a dissecting microscope. Each bar shows the mean ± SD of triplicate measurements. (D) Two-color analysis of IFN-{gamma} and IL-4 expression in CD4+ T cells stimulated by mature LCs. Cord blood CD4+ T cells (2x105) were cocultured with allogeneic, mature LCs (2x105) in the presence of anti-CD3 antibody. For expansion of the CD4+ T cells, IL-2 (20 IU/mL) was added to cultures on Day 4 of culture. After a 7-day culture, the cells were restimulated by PMA and ionomycin. Number of the recovered CD4+ T cells was 2.3 ± 0.4 x 106 (mean±SD in quadruplicate). Cytokine profiles in gated CD4+ T cells were studied by intracellular staining with anti-IFN-{gamma}-FITC and anti-IL-4-PE mAb. Analysis was done using a FACSCalibur flow cytometer. As a control, freshly isolated CD4+ T cells, which were stimulated by PMA and ionomycin, were used. Data are representative of three experiments (three different donors).

 
Chemotaxis of LCs generated from CD14+ monocytes
In terms of phenotype, LCs generated from CD14+ monocytes by Delta-1, GM-CSF, and TGF-ß1 differentiated into mature LCs in response to CD40L and TNF-{alpha}. To analyze the functional properties of these mature LCs, we analyzed their chemotaxis toward MIP-3{alpha} or MIP-3ß. MIP-3ß exhibited a substantial effect on the chemotaxis of mature LCs, and MIP-3{alpha} did not significantly induce the response (Fig. 5B) .

CD8+ T cell stimulation and CD4+ T cell polarization by LCs generated from CD14+ monocytes
The potential of mature LCs to stimulate CD8+ T cells was tested. We prepared mature LCs from CD14+ monocytes obtained from HLA-A*0201+ healthy donors and pulsed them with the modified Melan-A26–35 peptide (A27L), which stably binds to HLA-A*0201. After being cultured with these peptide-pulsed LCs, autologous CD8+ T cells were stimulated with peptide-pulsed T2 cells. The results presented in Figure 5C demonstrate that LCs with a mature phenotype efficiently induce IFN-{gamma}-producing CD8+ T cells in a peptide-specific manner. Next, to examine the influence of mature LCs on CD4+ T cell polarization, CD4+ T cells obtained from cord blood were cocultured with allogeneic, phenotypically mature LCs at a 1:1 ratio in the presence of anti-CD3 antibody. The CD4+ T cells were expanded with IL-2 and restimulated with PMA and ionomycin. The polarization of CD4+ T cells was evaluated at the single-cell level by analyzing IFN-{gamma}- or IL-4-producing cells using flow cytometry (Fig. 5D) . LCs with a mature phenotype preferentially polarized CD4+ T cells into IFN-{gamma}-producing cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The work presented here demonstrates that Notch ligand Delta-1, in cooperation with GM-CSF and TGF-ß1, supports the differentiation of human peripheral blood CD14+ monocytes into LCs. This indicates for the first time the involvement of Notch signaling in the development of human LCs from CD14+ monocytes. Our finding, that addition of a {gamma}-secretase inhibitor, capable of blocking Notch signaling, prevented the generation of LC-related antigen-expressing cells, supports this involvement of Notch signaling. Delta-1 as well as GM-CSF and TGF-ß1 are synthesized in the skin environment [30 ]. In this context, it is tempting to propose the hypothesis that monocytes, which have migrated to the skin, can be recruited to differentiate into LCs when they are exposed to a cytokine environment that consists of Delta-1, GM-CSF, and TGF-ß1. Delta-1 expression is detected at the basal layer of the epidermis, specifically by epidermal stem cells, which are surrounded by keratinocytes [30 ]. It is intriguing to speculate that chemokines produced by keratinocytes such as monocyte chemoattractant protein-1 could be involved in the direction of monocytes toward epidermal stem cells [45 ]. The Notch signaling plays a regulatory role in the cell differentiation processes in the early stages of hematopoiesis, such as lineage commitment of progenitor cells [18 , 19 , 46 ]. Our study shows that Notch signaling influences the lineage commitment of mature blood cells at the periphery. Canque et al. [47 ] reported that CD34+CD7+CD45RA+ cells differentiate into LCs. Delta-1 has been shown to support the expansion of CD34+CD7+CD45RA+ common lymphoid precursors from CD34+CD38– cells [21 ]. These observations, together with our data presented in this paper, suggest that Delta-1 plays a pivotal role in the development of LCs of lymphoid and myeloid origins.

Larregina et al. [15 ] defined CD14+ cells that reside in the human skin as immediate precursors for LCs. They claimed that these dermal-resident CD14+ cells were not of a monocyte-macrophage lineage. Further studies are necessary to clarify whether these CD14+ cells in the skin may be on the way from CD14+ monocytes toward LCs or represent a distinct lineage of cells. The cells cultured with GM-CSF, IL-4, and TGF-ß1 in our experiment are not comparable with LCs, generated from CD14+ monocytes with GM-CSF, IL-4, and TGF-ß1 in the previous study by Geissmann et al. [11 ]. At present, the basis for this difference is unclear. IL-15 in combination with GM-CSF was shown to induce DCs with features of LCs from CD14+ monocytes. These cells were similar to our cultured cells except for the expression of Birbeck granules [12 ]. These findings may reveal a complex network of multiple factors that regulate the development of LCs from monocytes. Guironnet et al. [13 ] reported that when CD14+ monocytes were cultured with GM-CSF and TGF-ß1, the cultured cells expressed Langerin and CCR6. Conversely, our data show that LC-related antigens were not induced in the cells cultured with GM-CSF and TGF-ß1. We have no likely explanation for the difference in phenotype. This difference does not appear to be significant, as part of their cultured cells expressed Langerin and CCR6. The mechanisms through which the Notch signaling promotes the development of LCs from monocytes have been currently unknown. Delta-1 was reported to inhibit the differentiation of monocytes into macrophages with GM-CSF [25 ]. We observed that in the presence of GM-CSF and TGF-ß1, CD14+ monocytes differentiate into macrophage-like cells but not into DCs or LCs. The inhibitory effect of Delta-1 on the differentiation of monocytes into macrophages appears to be one of the events in the generation of LCs from monocytes.

A recent work by Merad et al. [48 ], focusing on the life cycle of murine LCs, has described that LCs in the skin are replenished by skin-resident LC precursors under normal circumstances, whereas inflammatory stimuli, which are evoked by ultraviolet, lead to their replacement by circulating, blood-borne LC progenitors. As a variety of inflammatory signals results in migration of circulating monocytes into the peripheral tissues, it is possible that human monocytes in the inflamed skin may become LCs in response to Delta-1, GM-CSF, and TGF-ß1. However, it is unclear whether blood monocytes may contribute to a constant turnover of LCs under steady-state conditions in humans. The generation of LCs from human circulating CD14+ monocytes in our culture system did not appear to be associated with cell division, and murine LC precursors that were present in the skin and responsible for physiological replenishment of LCs underwent at least one division during their differentiation into LCs [48 ]. Distinct proliferative profiles of human peripheral blood monocytes and local murine LC precursors imply that murine local LC precursors are not progenies of blood monocytes.

An important issue is whether LCs generated in culture with Delta-1, GM-CSF, and TGF-ß1 retain their functions. The LCs displayed phagocytic activity and transmigrated through transwells in response to MIP-3{alpha}. These findings demonstrate that these LCs correspond to immature LCs. This is in accordance with our subsequent observation showing that in response to CD40L and TNF-{alpha}, the LCs exhibited phenotypic changes, which were manifested by up-regulation of HLA-ABC, HLA-DR, CD80, CD86, CD40, and CD54 and appearance of CD83. An unexpected finding was that mature LCs did not express CCR7 at a distinct level, although they showed chemotaxis in response to MIP-3ß. As the expression of CCR7 in mature LCs was evident, as detected by RT-PCR (data not shown), we presume that a small amount of CCR7 may facilitate chemotaxis of mature LCs across a gradient of MIP-3ß. We also observed that activated CD8+ T cells were induced by LCs, which were exposed to CD40L and TNF-{alpha}. CD4+ T cells are classified into two types of T helper cell types: Th1 and Th2 cells. Th1 cells produce IFN-{gamma}, which favors the priming and activation of cytotoxic T lymphocytes [49 ]. Of note is that the proportion of IFN-{gamma}-producing CD4+ T cells was high when cocultured with mature LCs, and IL-4-producing CD4+ T cells were found to be of low frequency. This indicates that LCs preferentially deliver a Th1-polarizing signal to CD4+ T cells. Taken together, these findings suggest that LCs generated from CD14+ monocytes in culture with Delta-1, GM-CSF, and TGF-ß1 have functional potentials.

In conclusion, human peripheral blood CD14+ monocytes can be instructed to differentiate into LCs in response to Notch ligand Delta-1, GM-CSF, and TGF-ß1, which are environmental cytokines in the skin. Definition of appropriate culture conditions allowing ex vivo generation of typical human LCs provides not only the opportunity for advances in the biology of LCs but also the possibility to apply LCs in a clinical setting.


   ACKNOWLEDGEMENTS
 
This work was supported in part by grants from the Japan Society for the Promotion of Science and Ministry of Health, Labour, and Welfare. N. H. and T. S. contributed equally to this work. We thank M. Ohara (Fukuoka) for critical comments and language assistance.

Received December 22, 2004; revised May 31, 2005; accepted June 29, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Silberberg-Sinakin, I., Thorbecke, G. J., Baer, R. L., Rosenthal, S. A., Berezowsky, V. (1976) Antigen-bearing Langerhans cells in skin, dermal lymphatics and in lymph nodes Cell. Immunol. 25,137-151[CrossRef][Medline]
  2. Schuler, G., Steinman, R. M. (1985) Murine epidermal Langerhans cells mature into potent immunostimulatory dendritic cells in vitro J. Exp. Med. 161,526-546[Abstract/Free Full Text]
  3. Inaba, K., Schuler, G., Witmer, M. D., Valinksy, J., Atassi, B., Steinman, R. M. (1986) Immunologic properties of purified epidermal Langerhans cells: distinct requirements for stimulation of unprimed and sensitized T lymphocytes J. Exp. Med. 164,605-613[Abstract/Free Full Text]
  4. Steinman, R. M. (1991) The dendritic cell system and its role in immunogenicity Annu. Rev. Immunol. 9,271-296[CrossRef][Medline]
  5. Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y-J., Pulendran, B., Palucka, K. (2000) Immunobiology of dendritic cells Annu. Rev. Immunol. 18,767-811[CrossRef][Medline]
  6. Volc-Platzer, B., Stingl, G., Wolff, K., Hinterberg, W., Schnedl, W. (1984) Cytogenetic identification of allogeneic epidermal Langerhans cells in a bone-marrow-graft recipient N. Engl. J. Med. 310,1123-1124[Medline]
  7. Emile, J. F., Haddad, E., Fraitag, S., Canioni, D., Fischer, A., Brousse, N. (1997) Detection of donor-derived Langerhans cells in MHC class II immunodeficient patients after allogeneic bone marrow transplantation Br. J. Haematol. 98,480-484[CrossRef][Medline]
  8. Reid, C. D. L., Fryer, P. R., Clifford, C., Kirk, A., Tikerpae, J., Knight, S. C. (1990) Identification of hematopoietic progenitors of macrophages and dendritic Langerhans cells (DL-CFU) in human bone marrow and peripheral blood Blood 76,1139-1149[Abstract/Free Full Text]
  9. Strunk, D., Rappersberger, K., Egger, C., Strobl, H., Kromer, E., Elbe, A., Maurer, D., Stingl, G. (1996) Generation of human dendritic cells/Langerhans cells from circulating CD34+ hematopoietic progenitor cells Blood 87,1292-1302[Abstract/Free Full Text]
  10. Strunk, D., Egger, C., Leitner, G., Hanau, D., Stingl, G. (1997) A skin homing molecule defines the Langerhans cell progenitor in human peripheral blood J. Exp. Med. 185,1131-1136[Abstract/Free Full Text]
  11. Geissmann, F., Prost, C., Monnet, J-P., Dy, M., Brousse, N., Hermine, O. (1998) Transforming growth factor ß1, in the presence of granulocyte/macrophage colony-stimulating factor and interleukin 4, induces differentiation of human peripheral blood monocytes into dendritic Langerhans cells J. Exp. Med. 187,961-966[Abstract/Free Full Text]
  12. Mohamadzadeh, M., Berard, F., Essert, G., Chalouni, C., Pulendran, B., Davoust, J., Bridges, G., Palucka, A. K., Banchereau, J. (2001) Interleukin 15 skews monocyte differentiation into dendritic cells with features of Langerhans cells J. Exp. Med. 194,1013-1019[Abstract/Free Full Text]
  13. Guironnet, G., Dezutter-Dambuyant, C., Vincent, C., Bechetoille, N., Schmitt, D., Peguet-Navarro, J. (2002) Antagonistic effects of IL-4 and TGF-ß1 on Langerhans cell-related antigen expression by human monocytes J. Leukoc. Biol. 71,845-853[Abstract/Free Full Text]
  14. Ito, T., Inaba, M., Inaba, K., Toki, J., Sogo, S., Iguchi, T., Adachi, Y., Yamaguchi, K., Amakawa, R., Valladeau, J., Saeland, S., Fukuhara, S., Ikehara, S. (1999) A CD1a+/CD11c+ subset of human blood dendritic cells is a direct precursor of Langerhans cells J. Immunol. 163,1409-1419[Abstract/Free Full Text]
  15. Larregina, A. T., Morelli, A. E., Spencer, L. A., Logar, A. J., Watkins, S. C., Thomson, A. W., Falo, L. D., Jr (2001) Dermal-resident CD14+ cells differentiate into Langerhans cells Nat. Immunol. 2,1151-1158[CrossRef][Medline]
  16. Artavanis-Tsakonas, S., Matsuno, K., Fortini, M. E. (1995) Notch signaling Science 268,225-232[Abstract/Free Full Text]
  17. Artavanis-Tsakonas, S., Rand, M. D., Lake, R. J. (1999) Notch signaling: cell fate control and signal integration in development Science 284,770-776[Abstract/Free Full Text]
  18. Milner, L. A., Bigas, A. (1999) Notch as a mediator of cell fate determination in hematopoiesis: evidence and speculation Blood 93,2431-2448[Free Full Text]
  19. Radtke, F., Wilson, A., Mancini, S. J. C., MacDonald, H. R. (2004) Notch regulation of lymphocyte development and function Nat. Immunol. 5,247-253[CrossRef][Medline]
  20. Karanu, F. N., Murdoch, B., Gallacher, L., Wu, D. M., Koremoto, M., Sakano, S., Bhatia, M. (2000) The Notch ligand Jagged-1 represents a novel growth factor of human hematopoietic stem cells J. Exp. Med. 192,1365-1372[Abstract/Free Full Text]
  21. Ohishi, K., Varnum-Finney, B., Bernstein, I. D. (2002) Delta-1 enhances marrow and thymus repopulating ability of human CD34+CD38– cord blood cells J. Clin. Invest. 110,1165-1174[CrossRef][Medline]
  22. Walker, L., Lynch, M., Silverman, S., Fraser, J., Boulter, J., Weinmaster, G., Gasson, J. C. (1999) The Notch/Jagged pathway inhibits proliferation of human hematopoietic progenitors in vitro Stem Cells 17,162-171[Abstract/Free Full Text]
  23. Masuya, M., Katayama, N., Hoshino, N., Nishikawa, H., Sakano, S., Araki, H., Mitani, H., Suzuki, H., Miyashita, H., Kobayashi, K., Nishii, K., Minami, N., Shiku, H. (2002) The soluble Notch ligand, Jagged-1, inhibits proliferation of CD34+ macrophage progenitors Int. J. Hematol. 75,269-276[Medline]
  24. Ohishi, K., Varnum-Finney, B., Flowers, D., Anasetti, C., Myerson, D., Bernstein, I. D. (2000) Monocytes express high amounts of Notch and undergo cytokine-specific apoptosis following interaction with the Notch ligand, Delta-1 Blood 95,2847-2854[Abstract/Free Full Text]
  25. Ohishi, K., Varnum-Finney, B., Serda, R. E., Anasetti, C., Bernstein, I. D. (2001) The Notch ligand, Delta-1, inhibits the differentiation of monocytes into macrophages but permits their differentiation into dendritic cells Blood 98,1402-1407[Abstract/Free Full Text]
  26. Sporn, M. B., Roberts, A. B. (1992) Transforming growth factor-ß: recent progress and new challenges J. Cell Biol. 119,1017-1021[Free Full Text]
  27. Luger, T. A., Bhardwaj, R. S., Grabbe, S., Schwarz, T. (1996) Regulation of the immune response by epidermal cytokines and neurohormones J. Dermatol. Sci. 13,5-10[CrossRef][Medline]
  28. Gasson, J. C. (1991) Molecular physiology of granulocyte-macrophage colony-stimulating factor Blood 77,1131-1145[Free Full Text]
  29. Pastore, S., Fanales-Belasio, E., Albanesi, C., Chinni, L. M., Giannetti, A., Girolomoni, G. (1997) Granulocyte macrophage colony-stimulating factor is overproduced by keratinocytes in atopic dermatitis: implications for sustained dendritic cell activation in the skin J. Clin. Invest. 99,3009-3017[Medline]
  30. Lowell, S., Jones, P., Le Roux, I., Dunne, J., Watt, F. M. (2000) Stimulation of human epidermal differentiation by delta-notch signaling at the boundaries of stem-cell clusters Curr. Biol. 10,491-500[CrossRef][Medline]
  31. Varnum-Finney, B., Purton, L. E., Yu, M., Brashem-Stein, C., Flowers, D., Staats, S., Moore, K. A., Le Roux, I., Mann, R., Gray, G., Artavanis-Tsakonas, S., Bernstein, I. D. (1998) The Notch ligand, Jagged-1, influences the development of primitive hematopoietic precursor cells Blood 91,4084-4091[Abstract/Free Full Text]
  32. Wolfe, M. S., Xia, W., Moore, C. L., Leatherwood, D. D., Ostaszewski, B., Rahmati, T., Donkor, I. O., Selkoe, D. J. (1999) Peptidomimetic probes and molecular modeling suggest that Alzheimer’s {gamma}-secretase is an intramembrane-cleaving aspartyl protease Biochemistry 38,4720-4727[CrossRef][Medline]
  33. Calvi, L. M., Adams, G. B., Weibrecht, K. W., Weber, J. M., Olson, D. P., Knight, M. C., Martin, R. P., Schipani, E., Divieti, P., Bringhurst, F. R., Milner, L. A., Kronenberg, H. M., Scadden, D. T. (2003) Osteoblastic cells regulate the haematopoietic stem cell niche Nature 425,841-846[CrossRef][Medline]
  34. Araki, H., Katayama, N., Mitani, H., Suzuki, H., Nishikawa, H., Masuya, M., Ikuta, Y., Hoshino, N., Miyashita, H., Nishii, K., Minami, N., Shiku, H. (2001) Efficient ex vivo generation of dendritic cells from CD14+ blood monocytes in the presence of human serum albumin for use in clinical vaccine trials Br. J. Haematol. 114,681-689[CrossRef][Medline]
  35. Davidson, D. J., Currie, A. J., Reid, G. S., Bowdish, D. M., MacDonald, K. L., Ma, R. C., Hancock, R. E., Speert, D. P. (2004) The cationic antimicrobial peptide LL-37 modulates dendritic cell differentiation and dendritic cell-induced T cell polarization J. Immunol. 172,1146-1156[Abstract/Free Full Text]
  36. Kovacs, B., Maus, M. V., Riley, J. L., Derimanov, G. S., Koretzky, G. A., June, C. H., Finkel, T. H. (2002) Human CD8+ T cells do not require the polarization of lipid rafts for activation and proliferation Proc. Natl. Acad. Sci. USA 99,15006-15011[Abstract/Free Full Text]
  37. Araki, H., Katayama, N., Yamashita, Y., Mano, H., Fujieda, A., Usui, E., Mitani, H., Ohishi, K., Nishii, K., Masuya, M., Minami, N., Nobori, T., Shiku, H. (2004) Reprogramming of human postmitotic neutrophils into macrophages by growth factors Blood 103,2973-2980[Abstract/Free Full Text]
  38. Nishii, K., Katayama, N., Miwa, H., Shikami, M., Usui, E., Masuya, M., Araki, H., Lorenzo, F., Ogawa, T., Kyo, T., Nasu, K., Shiku, H., Kita, K. (2002) Non-DNA-binding Ikaros isoform gene expressed in adult B-precursor acute lymphoblastic leukemia Leukemia 16,1285-1292[CrossRef][Medline]
  39. Dieu, M-C., Vanbervliet, B., Vicari, A., Bridon, J-M., Oldham, E., Ait-Yahia, S., Briere, F., Zlotnik, A., Lebecque, S., Caux, C. (1998) Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites J. Exp. Med. 188,373-386[Abstract/Free Full Text]
  40. Ikuta, Y., Katayama, N., Wang, L., Okugawa, T., Takahashi, Y., Schmitt, M., Gu, X., Watanabe, M., Akiyoshi, K., Nakamura, H., Kuribayashi, K., Sunamoto, J., Shiku, H. (2002) Presentation of a major histocompatibility complex class I-binding peptide by monocyte-derived dendritic cells incorporating hydrophobized polysaccharide-truncated HER2 protein complex: implications for a polyvalent immuno-cell therapy Blood 99,3717-3724[Abstract/Free Full Text]
  41. Romero, P., Gervois, N., Schneider, J., Escobar, P., Valmori, D., Pannetier, C., Steinle, A., Wolfel, T., Lienard, D., Brichard, V., van Pel, A., Jotereau, F., Cerottini, J. C. (1997) Cytolytic T lymphocyte recognition of the immunodominant HLA-A*0201- restricted Melan-A/MART-1 antigenic peptide in melanoma J. Immunol. 159,2366-2374[Abstract/Free Full Text]
  42. Salter, R. D., Cresswell, P. (1986) Impaired assembly and transport of HLA-A and -B antigens in a mutant TxB cell hybrid EMBO J. 5,943-949[Medline]
  43. Schroeter, E. H., Kisslinger, J. A., Kopan, R. (1998) Notch-1 signaling requires ligand-induced proteolytic release of intracellular domain Nature 393,382-386[CrossRef][Medline]
  44. Struhl, G., Adachi, A. (1998) Nuclear access and action of Notch in vivo Cell 93,649-660[CrossRef][Medline]
  45. Barker, J. N. W. N., Sarma, V., Mitra, R. S., Dixit, V. M., Nickoloff, B. J. (1990) Marked synergism between tumor necrosis factor-{alpha} and interferon-{gamma} in regulation of keratinocyte-derived adhesion molecules and chemotactic factors J. Clin. Invest. 85,605-608
  46. Radtke, F., Wilson, A., Ernst, B., MacDonald, H. R. (2002) The role of Notch signaling during hematopoietic lineage commitment Immunol. Rev. 187,65-74[CrossRef][Medline]
  47. Canque, B., Camus, S., Dalloul, A., Kahn, E., Yagello, M., Dezutter-Dambuyant, C., Schmitt, D., Schmitt, C., Gluckman, J. C. (2000) Characterization of dendritic cell differentiation pathways from cord blood CD34+CD7+CD45RA+ hematopoietic progenitor cells Blood 96,3748-3756[Abstract/Free Full Text]
  48. Merad, M., Manz, M. G., Karsunky, H., Wagers, A., Peters, W., Charo, I., Weissman, I. L., Cyster, J. G., Engleman, E. G. (2002) Langerhans cells renew in the skin throughout life under steady-state conditions Nat. Immunol. 3,1135-1141[CrossRef][Medline]
  49. Szabo, S. J., Sullivan, B. M., Peng, S. L., Glimcher, L. H. (2003) Molecular mechanisms regulating Th1 immune responses Annu. Rev. Immunol. 21,713-758[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
F. Edele, R. Molenaar, D. Gutle, J. C. Dudda, T. Jakob, B. Homey, R. Mebius, M. Hornef, and S. F. Martin
Cutting Edge: Instructive Role of Peripheral Tissue Cells in the Imprinting of T Cell Homing Receptor Patterns
J. Immunol., September 15, 2008; 181(6): 3745 - 3749.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
R. Shaykhiev and R. Bals
Interactions between epithelial cells and leukocytes in immunity and tissue homeostasis
J. Leukoc. Biol., July 1, 2007; 82(1): 1 - 15.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
P. Cheng, Y. Nefedova, C. A. Corzo, and D. I. Gabrilovich
Regulation of dendritic-cell differentiation by bone marrow stroma via different Notch ligands
Blood, January 15, 2007; 109(2): 507 - 515.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
K. G. Leong and A. Karsan
Recent insights into the role of Notch signaling in tumorigenesis
Blood, March 15, 2006; 107(6): 2223 - 2233.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.1204746v1
78/4/921    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hoshino, N.
Right arrow Articles by Shiku, H.
Right arrow