|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 1 (July 1), 1998:
pp. 118-128
Bifurcated Dendritic Cell Differentiation In Vitro From Murine
Lineage Phenotype-Negative c-kit+ Bone Marrow
Hematopoietic Progenitor Cells
By
Yi Zhang,
Akihisa Harada,
Jian-bin Wang,
Yan-yun Zhang,
Shin-ichi Hashimoto,
Makoto Naito, and
Kouji Matsushima
From the Department of Molecular Preventive Medicine and CREST,
School of Medicine, The University of Tokyo, Tokyo; and The Second
Department of Pathology, School of Medicine, Niigata University,
Niigata, Japan.
 |
ABSTRACT |
We have recently established the culture system to generate
dendritic cells (DCs) from murine
Lin c-kit+ bone marrow
hematopoietic progenitor cells (HPCs) in the presence of
granulocyte-macrophage colony-stimulating factor (GM-CSF) + stem cell
factor (SCF) + tumor necrosis factor- (TNF-). We present here
the identification of two DC precursor subsets originated from HPCs
with the phenotype of CD11b/dullCD11c+ and
CD11b+hiCD11c+ that develop independently
at early time points (days 4 to 6) in the same culture conditions. Both
of CD11b/dullCD11c+ and
CD11b+hiCD11c+ precursors could
differentiate at day 10 to 14 into
CD11b/dullCD11c+ mature DCs with typical
morphology, phenotype, and the ability to stimulate allogenic mixed
leukocyte reaction (MLR). However, the endocytic capacity of
fluorescein isothiocyanate-dextran was markedly reduced during the
differentiation. CD11b/dullCD11c+
precursors expressed high levels of Ia, CD86, CD40, and E-cadherin molecules, but not c-fms transcript, and mature DCs derived
from this precursor subset continue to express abundant E-cadherin antigen, a discernible marker for Langerhans cells. In contrast, CD11b+hiCD11c+ precursors expressed
c-fms mRNA, but low levels of Ia, CD86, and E-cadherin, whereas
CD40 was undetectable. CD11b/dullCD11c+
mature DCs differentiated from these precursors displayed abundant c-fms mRNA and nonspecific esterase activity.
Interestingly, CD11b+hiCD11c+
precursors, but not CD11b/dullCD11c+
precursors, may be bipotent cells that can be induced by M-CSF to
differentiate into macrophages. All of these results suggest that CD11b/dullCD11c+ and
CD11b+hiCD11c+ cells are distinct DC
precursors derived from Linc-kit+ HPCs,
which differentiate into mature DCs through bifurcated and independent
DC differentiation pathways.
| |
INTRODUCTION |
DENDRITIC CELLS (DCs) that have the
capacity to initiate immune responses as professional
antigen-presenting cells consist of heterogeneous cell populations and
are distributed in nonlymphoid as well as lymphoid
organs.1,2 Phenotypic differences between murine thymic and
splenic DCs have been based on the expression of BP-1 and Thy-1
antigens that are exclusively expressed on thymic DCs but not splenic
ones.3-7 In the skin, epithelial Langerhans cells (LCs)
express Birbeck granules+ and E-cadherin+,
whereas dermal dendrocytes do not have Birbeck granules, but express
factor XIIIa+.8,9 Functionally different
subsets of DCs have been also identified based on phenotypic criteria.
In human peripheral blood (PB), the CD11c and
CD83 subsets have been shown to be immature DCs, whereas
CD11c+ and CD83+ markers identify mature DCs
displaying considerable T-cell-stimulatory capacity.10,11
Murine thymic DCs can be further subdivided into major
histocompatibility complex (MHC) class IIlo and MHC class
IIhi DC subsets which represent immature and mature DCs,
respectively.12 Moreover, DCs with CD8 antigen induce
apoptosis of activated T cells in contrast to CD8
DCs.13 Taken together, all these findings indicate that DCs are not only phenotypically but also functionally heterogeneous.
DCs generated from cultured hematopoietic progenitor cells (HPCs) in
vitro also consist of heterogeneous populations. Human cord blood
CD34+ HPCs have been reported to differentiate in vitro
into at least two DC precursor subsets by distinct pathways based on
their CD1a and CD14 expression.14
CD1a+CD14c-fms
precursors can give rise to Birbeck granule+ DCs resembling
epithelial LCs, whereas
CD1aCD14+c-fms+
precursors can generate Birbeck granulefactor
XIIIa+ blood or dermal dendrocytic DCs as well as
macrophages. The most striking difference is the unique capacity of
CD14+ precursor-derived DCs to induce naive B cells to
differentiate into IgM-secreting cells.15 These findings
suggest that DC progenitor cells can differentiate into functionally
distinct DC subtypes and that different DC precursor subsets may exist
for DCs distributed in various tissues.
Multiple DC subpopulations have been identified in vivo in the spleen
of Flt3 ligand (Flt3L)-treated mice based on the expression of CD11c,
CD11b, and CD8.16,17
CD11bdullCD11c+ and
CD11bCD11c+ DC subsets abundantly express
CD8, whereas CD11bbrightCD11c+ DCs are
negative for this molecule. These three DC subsets all localize to
different sites in the spleen of Flt-3L-treated mice.17 Interestingly, in vivo treatment with Flt3L only induced an increase in
the CD11bCD11c+ DC subset, but not others,
in the thymus,17 suggesting that distinct differentiation
pathways also exist in Flt3L-treated mice. However, it remains unknown
whether these multiple DC subsets might differentiate from a common
precursor or simply represent an immature stage in the development of
DC population. We have recently established a culture system to
generate DCs from murine bone marrow (BM)
Linc-kit+ HPCs in the presence of
granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell
factor (SCF), and tumor necrosis factor- (TNF-).7
Using the same culture system, here we investigate the cellular basis
of DC differentiation from
Linc-kit+ HPCs, and find that
CD11b/dullCD11c+ and
CD11b+hiCD11c+ DC precursors represent two
distinct intermediate-stage cell subsets with the capacity to
differentiate into mature DCs.
| |
MATERIALS AND METHODS |
Cytokines and antibodies.
Recombinant murine SCF and GM-CSF were kindly provided by Kirin Brewery
Co (Tokyo, Japan) and Dr T. Sudo (Basic Research Institute of Toray Co,
Kanagawa, Japan), respectively. M-CSF was kindly provided by Morinaga
Milk Industry Cooperation (Morinaga, Japan). Mouse TNF-
was produced as described previously.18 Endotoxin was not
detectable in these cytokine preparations using a Toxicolor assay kit
(Seikagaku-Kogyo, Tokyo, Japan). These cytokines were used at the
optimal concentrations as previously described.7 An
anti-c-kit antibody (ACK-2) was kindly provided by Dr T. Sudo (Toray, Kanagawa, Japan) and conjugated with biotin by using an NHS-Biotin kit (Pharmacia-Biotech, Uppsala, Sweden) according to the
manufacturer's instructions.19 A rat monoclonal antibody (MoAb) to murine dendritic cell marker, DEC-205 (NLDC145), was a
generous gift of Dr R.M. Steinman (Rockefeller University, New York,
NY).20,21 MoAb to mouse E-cadherin was purchased from Dainipon Pharmaceutical Co (Tokyo, Japan). Other MoAbs and reagents used for immunostaining were obtained from PharMingen (San Diego, CA),
unless otherwise indicated.
Mice.
C57BL/6 and Balb/c mice were obtained from Kurea Animal Co (Tokyo,
Japan) and maintained under pathogen-free conditions in the Animal
Facility of Department of Molecular Preventive Medicine, School of
Medicine, the University of Tokyo (Tokyo, Japan). All animal
experiments complied with the standards set out in the Guideline for
Care and Use of Laboratory Animals of the University of Tokyo.
Suspension culture of
Linc-kit+ HPCs.
BM cells were obtained by aspirating femurs and tibiae of 8- to
10-week-old female mice. Linc-kit+
HPCs were isolated from nonadherent BM mononuclear cells (MNCs) using
an EPICS ELITE cell sorter (Coulter Electronics, Hialeah, FL) as
previously described.7,22,23 In brief, nonadherent MNCs
were stained with an indirect staining composed of biotin-conjugated anti-c-kit MoAb and phycoerythrin (PE)-labeled streptavidin
followed by a set of fluorescein isothiocyanate (FITC)-labeled MoAbs to CD3 (145-2C11), CD4 (H129.19), CD8 (53-6.7), B220 (RA3-6B2), Gr-1 (Ly-6G), CD11a (2D7), and CD11b (M1/70). The contamination by other
types of cells in these preparations was consistently less than 0.5%
as shown by an immunofluorescence analysis.
Purified Linc-kit+ HPCs were
incubated as previously described7 at a cell concentration
of 1 × 104 cells/mL in Iscove's modified Dulbecco's
medium (IMDM; GIBCO, Rockville, MD) supplemented with 10% fetal bovine
serum (FBS), 5 × 105 mol/L 2-mercaptoethanol,
penicillin G (100 U/mL), and streptomycin (100 µg/mL) in the presence
of GM-CSF + SCF + TNF-. Optimal conditions were maintained by
splitting these cultures at day 4 with medium exchange containing fresh
cytokines. For most experiments cells were collected after 6 days of
culture for cell sorting.
Isolation of CD11b/dullCD11c+
and CD11b+hiCD11c+ DC precursors by a
cell sorter.
After 6 days of culture in the presence of GM-CSF, SCF, and TNF-,
cells were collected, labeled with FITC-conjugated anti-CD11b and
PE-conjugated anti-CD11c (HL3), and sorted into
CD11b/dullCD11c+ and
CD11b+hiCD11c+ cell subsets. In some
experiments, the CD11bCD11c cell fraction
was also sorted. All the staining and sorting procedures were performed
in the presence of 1 mmol/L EDTA to avoid cell aggregation. Reanalysis
of the sorted populations showed a purity higher than 98%. Sorted
cells were routinely incubated in medium containing GM-CSF + TNF- or
M-CSF for an additional 5 to 8 days. The cultured cells were collected
between days 10 and 14.
Immunofluorescence analysis.
Immunofluorescence analyses were performed as previously
described.7,22,23 In three-color analyses, 4 × 105 cells were incubated with biotinylated hamster
anti-CD11c MoAbs and rat anti-CD11b MoAbs, followed by staining with
Cy-Chrome (CY)-labeled streptavidin and PE-conjugated goat anti-rat IgG (Fab )2 antibody. The cells were then stained with
various FITC-conjugated MoAbs. In some experiments, the cells were
first stained with rat anti-E-cadherin MoAb and biotinylated
anti-CD11c, followed by staining with PE-conjugated goat anti-rat
IgG(Fab)2 antibody and CY-labeled streptavidin, and then
stained with FITC-conjugated anti-CD11b. In other experiments, the
cells were first stained with biotinylated antibodies and revealed by
CY-conjugated streptavidin, followed by staining with PE-labeled
anti-CD11c and FITC-conjugated anti-CD11b. The instrument compensation
was set in each experiment using single-color and/or two-color
stained samples.
Reverse transcription-polymerase chain reaction (RT-PCR).
Total RNAs were extracted from 1 × 105 indicated cells
using RNAzol B (Biotex Laboratories Inc, Houston, TX), according to the
manufacturer's instructions. First-strand cDNA was synthesized in a
25-µL reaction volume using an RT-PCR kit (Takara Shuzo, Kyoto,
Japan) with random primers. Thereafter, cDNA was amplified for 25 cycles consisting of 94°C for 30 seconds, 57°C for 1 minute, and
72°C for 1.5 minutes with the c-fms-specific oligonucleotide primers (5-CCAGAACTGGTTGTAGAGCC-3 and 5-CAGCTTGCTAGGCTCCAATT-3), which specifically result in a 500-bp cDNA encoding
c-fms.24 As a control, mouse  -actin transcript
was amplified in parallel as previously described.7 The PCR
products were fractionated on 1.5% agarose gel and visualized by
ethidium bromide staining.
Endocytosis.
The endocytosis experiments were performed as previously
reported.25 In brief, cells were incubated with 0.1 mg/mL
FITC-Dextran (FITC-DX; 4,000 daltons; Sigma Chemical Co, St Louis, MO)
at 37°C or 0°C for 60 minutes. Uptake was stopped by
adding ice-cold phosphate-buffered saline (PBS) containing 5%
bovine serum albumin (BSA) and 0.02% sodium azide. After staining with
biotinylated anti-CD11c and rat anti-CD11b MoAbs, the cells were
further incubated with CY-labeled streptavidin and PE-conjugated
goat anti-rat IgG (Fab)2 antibody followed by
flow cytometric analysis.
Mixed leukocyte reaction (MLR).
Splenic MNCs were prepared from allogenic mice (Balb/c) as described
previously.7 The adherent cells were first removed by
incubating them at 37°C for 60 minutes in IMDM containing 10% fetal
calf serum (FCS). To obtain highly purified T cells, the nonadherent
splenic MNCs were incubated with rat anti-B220 and mouse anti-Ia MoAbs
followed by staining with anti-rat IgG and anti-mouse IgG conjugated
magnetic beads to deplete B220+ and Ia+ cells
using Dynal-beads (Dynal, Oslo, Norway). After treatment with mitomycin
C (MMC; 15 µg/mL),26 the indicated stimulator cells (from
100 to 3 × 104 cells) were added to T cells
(3 × 105) in each well of 96-well round-bottomed
microtest tissue-culture plates (Nunc, Roskilde, Denmark). After
incubating at 37°C for 4 to 5 days, cell proliferation was determined
using 3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyltetrazolium bromide
(MTT; Sigma Chemical Co). In brief, 15 µL of MTT (5 mg/mL in PBS) was
added into each well and the plates were incubated at 37°C for an
additional 4 hours. The resultant absorbance at 550 nm was read by a
microplate immunoreader.
Electron microscopy.
Cultured cells sampled after 7 and 14 days of culture were fixed in 2%
glutaraldehyde, postfixed in 1% osmium tetroxide, and embedded in Epon
812 (E. Fullan, Inc, Latham, NY). Ultrathin sections were then cut and
stained with uranyl acetate and lead citrate, and examined with an
electron microscope, HITACHI H-800 (HITACHI, Tokyo,
Japan).27
Statistical analysis.
Differences were evaluated using the Student's t-test.
P values of <.05 were considered to be statistically
significant.
| |
RESULTS |
Differentiation of two DC precursor subsets from
Linc-kit+ HPCs.
To elucidate the cellular basis of the development of DC, we followed
the kinetics of CD11b and CD11c expression during differentiation of
murine Linc-kit+ HPC stimulated
with GM-CSF + SCF + TNF-. At day 0, murine
Linc-kit+ HPCs did not express
CD11b or CD11c (Fig 1A). However, two
cell populations characterized by the expression of
CD11b/dullCD11c+ and
CD11b+hiCD11c+ emerged independently in the
cultures at day 4. At day 6, a distinct population of
CD11b/dullCD11c+ cells could be
identified (7.2% ± 1.2%) which increased rapidly thereafter and
reached the maximum levels by days 10 to 14. Although CD11b+hiCD11c+ cells (22.9% ± 3.5%)
were usually dominant over the
CD11b/dullCD11c+ cells in the
cultures at day 6, they did not increase thereafter during more prolonged culture periods (Fig 1A), implying that CD11b+hiCD11c+ cells may contribute to
the later generation of CD11b/dullCD11c+
cells in the cultures. To examine this possibility, the three cell
populations CD11b/dullCD11c+,
CD11b+hiCD11c+, and CD11b
CD11c were routinely sorted at day 6 (Fig 1B) and
cultured in the presence of GM-CSF + TNF- for an additional 5 to 8 days. Most of the CD11b+hiCD11c+ cells could
differentiate into cells with the
CD11b/dullCD11c+ (51.0% ± 10.4%;
n = 15) phenotype at days 12 to 14. In contrast, CD11b/dullCD11c+-derived cells did not
change their phenotype at days 10 to 12 (81.7 ± 11.2%, n = 15)
and did not revert into a CD11b+hi phenotype.
Interestingly, the CD11bCD11c cell
population generated both of
CD11b/dullCD11c+ (8.4% ± 2.1%,
n = 4) and CD11b+hiCD11c+
(25.1% ± 3.4%, n = 4) cells at a similar rate and extent
as Linc-kit+ HPC cultures at day 6 (Fig 1B). These results suggest that
Linc-kit+ HPC may generate
CD11b/dullCD11c+ cells through at least two
independent differentiation pathways: one may directly differentiate
from Linc-kit+ HPCs at the early
stage, while another one is an intermediate stage for
CD11b+hiCD11c+ cells at the latter stage of
culture.
View larger version (25K):
[in this window]
[in a new window]
| Fig 1.
Generation of DC precursors and DCs from murine
Linc-kit+ HPCs stimulated with
GM-CSF + TNF-. (A) Murine
Linc-kit+ HPCs were cultured in
the presence of GM-CSF + SCF + TNF- for 6 to 7 days and then in
the presence of GM-CSF + TNF- for another 7 days. At the indicated
timepoint, independent aliquots of cells were recovered and processed
for analyses of CD11b and CD11c expression by using double-color
immunostaining with anti-CD11b-FITC and anti-CD11c-PE. The data
represent mean value ± SD of percentage of the two subpopulations
observed in more than five experiments. *P < .05 significance
as compared with CD11b/dullCD11c+ cells at
the indicated timepoint. (B) The cells were routinely sorted from
cultures of GM-CSF + SCF + TNF--stimulated murine Linc-kit+ HPCs at day 6 into
CD11b/dullCD11c+,
CD11b+hiCD11c+, and
CD11bCD11c cell populations using EPICS
ELITE cell sorter (middle panel). The sorted cells were cultured again
in the presence of GM-CSF + TNF- for an additional 5 to 8 days and
reanalyzed for the expression of CD11b and CD11c by double-color immune
staining (right panel). Quads were set up on the isotype-matched
control dot plot and the results are representative of more than 15 experiments.
|
|
To further characterize the phenotype of
CD11b/dullCD11c+ and
CD11b+hiCD11c+ cells generated in the cultures
at day 6, three-color immunofluorescence analyses were performed. These
showed that both of the populations were negative for CD4 and B220
(data not shown), CD8, and Gr-1 (Fig 2).
Although both of the subsets expressed comparable levels of Thy-1,
CD11a, and CD32/16, CD11b/dullCD11c+ cells
expressed much higher levels of Ia, CD86, E-cadherin, and DEC-205 than
CD11b+hiCD11c+ cells. Interestingly,
CD11b/dullCD11c+ cells also expressed high
levels of CD40 that were undetectable on
CD11b+hiCD11c+ cells at this time point (Fig
2). Taken together, these data would indicate that the two populations
differ phenotypically from each other in the expression of a number of
surface markers.
View larger version (21K):
[in this window]
[in a new window]
| Fig 2.
The CD11b/dullCD11c+ and
CD11b+hiCD11c+ DC precursors display a
different immunophenotype. Murine
Linc-kit+ HPCs were cultured in
presence of GM-CSF + SCF + TNF- for 6 days. The phenotype of the
CD11b/dullCD11c+ and
CD11b+hiCD11c+ precursors was determined by
three-color immune staining using uncoupled test MoAbs shown by
PE-conjugated anti-rat Ig and biotinylated anti-CD11c MoAb shown by
CY-conjugated streptavidin, and the third color was shown by
FITC-conjugated anti-CD11b MoAb. In some experiments FITC-conjugated
anti-CD11b and PE-conjugated anti-CD11c MoAbs were used, while the test
biotinylated MoAbs were shown by CY-conjugated streptavidin. In some
experiments, uncoupled CD11b and biotinylated CD11c were shown by
PE-conjugated anti-rat IgG and CY-conjugated streptavidin and finally
FITC-conjugated test MoAbs. Histograms shown in the figures are gated
on CD11b/dullCD11c+ and
CD11b+hiCD11c+ DC precursors. Solid and
dotted lines indicate the immunofluoresecence intensity of cells
stained with a control and the test antibodies, respectively.
Representative results from three or more independent experiments are
shown.
|
|
Because both CD11b/dullCD11c+ and
CD11b+hiCD11c+ cells can differentiate
into cells of the CD11b/dullCD11c+
phenotype, a previously demonstrated DC specific
phenotype,3-7,16,17 we therefore designated these two cell
populations sorted from Linc-kit+ HPC cultures at day 6 as
CD11b/dullCD11c+ and
CD11b+hiCD11c+ DC precursors, respectively.
Both CD11b/dullCD11c+ and
CD11b+hiCD11c+ DC precursors can
differentiate into mature phenotypically distinct DC-like cells.
To better understand the phenotypic differences between the two DC
precursors and their derived mature DCs,
CD11b/dullCD11c+ and
CD11b+hiCD11c+ DC precursors were routinely
sorted at day 6 and recultured in the presence of GM-CSF + TNF-.
Stimulation with GM-CSF + TNF- could not again induce proliferation
of CD11b/dullCD11c+ and
CD11b+hiCD11c+ DC precursors (data not shown).
Upon culture with GM-CSF + TNF-, CD11b/dullCD11c+ DC precursors developed
into a relatively homogeneous, plastic nonadherent cell population that
were irregular in shape and congregated into large aggregates of DC
surrounded by cells with long spiny processes at days 10 to 12 (Fig 3A
through C). In contrast, most CD11b+hiCD11c+ DC precursors appeared to be
adherent cells, medium to large in size that could form large DC
aggregates at later time points of days 12 to 14 under the same culture
conditions (Fig 3D and E). These cells could be easily detached and had
the morphological characteristics of DC-like cells (Fig 3F).
View larger version (127K):
[in this window]
[in a new window]
| Fig 3.
The day 6 DC precursors differentiate into cells
displaying a dendritic cell morphology at days 10 to 14. CD11b/dullCD11c+ and
CD11b+hiCD11c+ precursors were sorted from
murine Linc-kit+ HPC
cultures stimulated with GM-CSF + SCF + TNF- for 6 days. A
phase contrast microscopical observation (A, D, G, and H) and May-Grünwald-Giemsa staining (B, C, E, F, and I) were performed on sorted CD11b/dullCD11c+ and
CD11b+hiCD11c+ precursors or after
culture for 5 to 8 additional days in the presence of GMCSF + TNF- or M-CSF, respectively. (A and C)
CD11b/dullCD11c+ precursors cultured
for 5 to 6 days in the presence of GM-CSF + TNF-; (B)
sorted CD11b/dullCD11c+ precursors;
(D and F) CD11b+hiCD11c+
precursors cultured for additional 5 to 8 days in presence of GM-CSF + TNF-; (E) sorted
CD11b+hiCD11c+ precursors; (G)
CD11b/dullCD11c+ precursors cultured in
the presence of M-CSF for 3 days; (H and I) cultured
CD11b+hiCD11c+ precursors in the
presence of M-CSF for 5 to 8 days. Original magnifications: (A) ×200;
(D, G, and H) ×160; (B, C, E, F, and I) ×400.
|
|
During the differentiation of these two DC precursors, c-fms
was exclusively expressed by CD11b+hiCD11c+ DC
precursors and their mature offspring, but not by
CD11b/dullCD11c+ DC precursors and their
mature CD11b/dullCD11c+ offspring (Fig
4A). Interestingly, most of the
CD11b+hiCD11c+ DC precursors (Fig 4D) and
their mature offspring (Fig 4E) also displayed nonspecific esterase
activity in contrast with the
CD11b/dullCD11c+ DC precursors (Fig
4B) and their mature offspring (Fig 4C) in which the nonspecific
esterase activity was undetectable.
View larger version (99K):
[in this window]
[in a new window]
| Fig 4.
Expression of c-fms mRNA and nonspecific esterase
activity in CD11b/dullCD11c+,
CD11b+hiCD11c+ DC precursors, and their
derived DCs. (A) Expression of c-fms mRNA was examined in the
indicated cells using RT-PCR. Total RNAs were extracted from 1 × 105 indicated cells. The -actin transcripts were used as
control. Lane 1, 1-kb DNA ladder; 2, Linc-kit+ HPC; 3, CD11b/dullCD11c+ precursor; 4, CD11b/dullCD11c+ mature DC
derived from
CD11b/dullCD11c+ precursors; 5, CD11b+hiCD11c+ precursor; 6, CD11b/dullCD11c+ mature DC derived from
CD11b+hiCD11c+ precursors; 7, macrophage derived from M-CSF-induced
CD11b+hiCD11c+ DC precursors. (B through F)
The cultured cells were sorted and processed for nonspecific
esterase staining. (B) CD11b/dullCD11c+
precursors; (C) CD11b/dullCD11c+
precursor-derived CD11b/dullCD11c+
mature DCs; (D)
CD11b+hiCD11c+ DC precursors; (E)
CD11b+hiCD11c+ DC precursor-derived
CD11b/dullCD11c+ mature DCs; (F)
macrophages derived from M-CSF-induced
CD11b+hiCD11c+ DC precursors. Original
magnification ×400.
|
|
Ultrastructural observation showed that both
CD11b/dullCD11c+ (Fig
5A) and
CD11b+hiCD11c+ DC precursors (Fig 5B) were
small and mostly round in shape. They contained mitochondria and rough
endoplasmic in the cytoplasm and projected a few short cytoplasmic
processes. After culture for an additional 5 to 8 days, the cells
became larger and showed well-developed Golgi apparatus, mitochondria,
rough endoplasmic reticulum (RER), and a few small lysosomes. The cells
also developed abundant long cytoplasmic processes, a tubulovesicular
system, vesicles, and multivesicular bodies near the nucleus,
particularly adjacent to the Golgi apparatus in the
cytoplasmic (Fig 5C and D). There were no
significant morphological differences between CD11b/dullCD11c+ DC precursor-derived mature
DCs and CD11b+hiCD11c+ precursor-derived ones.
View larger version (98K):
[in this window]
[in a new window]
View larger version (119K):
[in this window]
[in a new window]
View larger version (112K):
[in this window]
[in a new window]
View larger version (122K):
[in this window]
[in a new window]
| Fig 5.
Electron microscopy of typical
CD11b/dullCD11c+ precursor and its derived
CD11b/dullCD11c+ mature DCs. The
CD11b/dullCD11c+ precursors and its
derived CD11b/dullCD11c+ mature DCs were
sorted at culture day 6 and day 12, respectively, and processed for
electron microscopy staining as described in Materials and Methods. (A)
A representative of CD11b/dullCD11c+
precursor sorted at day 6. Original magnification ×5,300. (B) A
representative of CD11b/dullCD11c+
precursor-derived CD11b/dullCD11c+ mature
DC sorted at day 12. Original magnification ×5,300. (C) A
representative of CD11b+hiCD11c+ precursor
sorted at day 6. Original magnification ×9,600. (D) A representative
of CD11b+hiCD11c+ precursor-derived mature
DC with the phenotype of CD11b/dullCD11c+
sorted at day 12. Original magnification ×9,600.
|
|
To characterize the immunophenotype of the mature DCs differentiated
from the two DC precursor subsets, three-color immunofluorescence analyses were performed. As shown in Fig
6,CD11b/dullCD11c+ mature DCs derived from
either CD11b/dullCD11c+ or
CD11b+hiCD11c+ DC precursors expressed higher
levels of Ia, CD86, CD40, and DEC-205, characteristic of active mature
DC. However, E-cadherin antigen, a discernible marker for
LCs,28 was exclusively expressed only on
CD11b/dullCD11c+ DC precursor-derived mature
DCs but not by CD11b+hiCD11c+ precursor-derived
ones. All these results indicate that
CD11b+hiCD11c+ and
CD11b/dullCD11c+ DC precursors and their
derived mature DCs share a combination of different phenotypes and
cannot be converted by each other, even though their
mature offspring have the common phenotype of CD11b/dullCD11c+.
View larger version (28K):
[in this window]
[in a new window]
| Fig 6.
The day 6 DC precursors differentiate into cells with
typical DC markers. CD11b/dullCD11c+
and CD11b+hiCD11c+ precursors were sorted
from murine Linc-kit+ HPC
cultures stimulated with GM-CSF + SCF + TNF- for 6 days and were
cultured in the presence of GMCSF + TNF- for an additional 5 to 8 days. At days 12 to 14, the phenotype of the
CD11b/dullCD11c+ DC-like cells
derived from either CD11b/dullCD11c+ or
CD11b+hiCD11c+ precursors was determined
using three-color analyses as described in the legend to Fig 2.
|
|
Mature DCs derived from
CD11b/dullCD11c+ and
CD11b+hiCD11c+ DC precursors each
stimulate allogenic MLR.
As examined by allogenic MLR,
CD11b/dullCD11c+ DC precursors,
but not CD11b+hiCD11c+ ones, could
effectively enhance allogenic MLR, suggesting that CD11b/dullCD11c+ cells may
be functionally active in presenting antigen as well as stimulating
T-cell proliferation (Fig 7A). However,
after culture in the presence of GM-CSF + TNF- for an additional 5 to 8 days, CD11b/dullCD11c+ mature DCs
derived from either CD11b+hiCD11c+ or
CD11b/dullCD11c+ precursors each became
equally potent in their capacity to stimulate in vitro T-lymphocyte
proliferation (Fig 7B). In comparison with CD11b/dullCD11c+ DCs matured at days
12 to 14, CD11b/dullCD11c+ DC precursors
were less potent in enhancing allogenic MLR, indicating that both
CD11b/dullCD11c+ and
CD11b+hiCD11c+ DC precursors can differentiate
into functional mature DCs in response to GM-CSF + TNF- at days 12 to 14, while CD11b/dullCD11c+ DC precursors
are still functionally immature at day 6, even though they express
active markers of DC.
View larger version (24K):
[in this window]
[in a new window]
| Fig 7.
The capacity of the cultured cells to enhance allogenic
MLR. Allogenic MLR was performed using purified T cells
(3 × 105 cells per well in 96 round-well plates) as
responder cells. (A) The day 6 sorted
CD11b/dullCD11c+,
CD11b+hiCD11c+, and
CD11bCD11c precursors, and unfractionated
cells from Linc-kit+ HPC
cultures were treated with MMC and used as stimulators at the indicated
cell number. (B) Sorted mature
CD11b/dullCD11c+ mature DCs derived from
CD11b/dullCD11c+,
CD11b+hiCD11c+ DC precursors at days 12 to
14 and macrophages derived from M-CSF-induced CD11b+hiCD11c+ DC precursors were used as
stimulator cells at the indicated cell number. The proliferation of T
cells was measured by MTT assay after 4 days of culture. Results are
expressed as mean ± 1 SD of triplicate cultures. Results of each
panel are representative of three independent experiments.
|
|
Decrease in endocytic ability during
CD11b/dullCD11c+ and
CD11b+hiCD11c+ DC precursor
maturation.
During maturation, DCs gradually lose their endocytic
ability.25,29,30 As shown in Fig
8, either
CD11b/dullCD11c+ or
CD11b+hiCD11c+ precursors could efficiently
take up FITC-DX at 37°C, but this was blocked by incubating them at
0°C. This capacity was significantly reduced when these cells
differentiated into
CD11b/dullCD11c+ mature DCs
induced by GM-CSF + TNF- by days 12 to 14. These data suggest that
micropinocytosis is highly reduced during maturation and
differentiation of DC precursors.
View larger version (24K):
[in this window]
[in a new window]
| Fig 8.
FITC-DX uptake by
CD11b/dullCD11c+ precursors ( ) and
CD11b+hiCD11c+ DC precursors () at day 6 and their derived CD11b/dullCD11c+ mature
DCs at days 12 to 14. Cells were first exposed to 0.1 mg/mL of FITC-DX
at 0°C and 37°C, respectively, for 60 minutes. After washing twice,
the cells were stained with rat-anti-mouse CD11b and biotinylated
hamster-anti-mouse CD11c and then shown by PE-conjugated anti-rat Ig
and CY-conjugated-streptavidin. A three-color immunofluorescence
analysis was performed to show the capacity of FITC-DX uptake by these
cells as indicated. *P < .01 significance compared with
CD11b/dullCD11c+ ( ) and
CD11b+hiCD11c+ ( ) DC precursor-derived
mature DCs. Results are representative of three experiments.
|
|
CD11b+hiCD11c+ but not
CD11b/dullCD11c+ DC precursors
differentiate into macrophage when stimulated with M-CSF.
Upon stimulating with M-CSF for an additional 5 to 8 days, all of the
CD11b+hiCD11c+ precursors differentiated
uniformly into macrophages with numerous vacuoles and nonspecific
esterase activity (Fig 3H and I, Fig 4F). These M-CSF-induced cells
expressed moderate to high levels of CD11b but not Ia, CD11c, CD86, or
DEC-205 molecules (Fig 9A) and were
incapable of stimulating allogeneic MLR (Fig 7B). In contrast, M-CSF
did not induce CD11b/dullCD11c+ DC
precursors to develop into macrophages and all of them died within 3 days in the cultures (Fig 3G). This is consistent with the fact that
c-fms transcripts were selectively expressed in CD11b+hiCD11c+ DC precursors but not in
CD11b/dullCD11c+ precursors (Fig 4A). These
results indicate that CD11b+hiCD11c+ precursors
may have dual potential to differentiate into either mature DCs or
macrophages depending on the supplemented growth factors, whereas
CD11b/dullCD11c+ precursors have already
committed to DC lineage, as illustrated in Fig 9B.
View larger version (20K):
[in this window]
[in a new window]
| Fig 9.
The dual differentiation potential of
CD11b+hiCD11c+ precursors. (A) Day 6 sorted
CD11b+hiCD11c+ DC precursors from
Linc-kit+ HPCs were cultured in
presence of GM-CSF + TNF- and M-CSF, respectively, for 5 to 8 additional days. At days 12 to 14, the phenotypes of cells were
reanalyzed using double-color immunofluorecence as indicated. For
double-color immunostaining, PE-conjugated anti-Ia was used, whereas
biotinylated anti-CD11c and CD86 were shown by FITC-conjugated
streptavidin and anti-DEC-205 was shown by FITC-labeled anti-rat IgG.
The representative expression shown here is one of more than five
experiments. (B) A schematic DC differentiation model in vitro from
Linc-kit+ HPCs.
|
|
| |
DISCUSSION |
Previous studies have shown the generation of heterogeneous mature DCs
in the cultures of mouse BM hematopoietic cells.7,27,31,32 However, it remains to be established whether the heterogeneous DC
subsets may differentiate from a distinct precursor and/or the
same precursor committed to DC at a different maturation stage. The
present investigation shows that
Linc-kit+ HPCs generate mature DCs
in vitro in response to GM-CSF + TNF- through the bifurcated DC
differentiation pathways CD11b/dullCD11c+
and CD11b+hiCD11c+ DC precursors, respectively
(Fig 9B). The CD11b/dullCD11c+ DC precursors
expressed the high levels of CD40, Ia, and CD86. Morphologically, the
CD11b/dullCD11c+ DC precursors were small
and mostly round in shape with less cytoplasmic projections than mature
DCs as shown by electron microscopy observation. These cells
differentiated into mature DCs, but not other myeloid cells, and became
large in size with abundant long cytoplasmic processes and
multivesicules. During maturation, the endocytic capacity was reduced
while the capability of stimulating the proliferation of allogeneic T
cells was significantly enhanced. All these features suggest that
CD11b/dull CD11c+ DC precursors are
committed DC precursors at a functionally immature stage, which makes
them distinguishable from CD11b/dullCD11c+
mature DCs.
In contrast, CD11b+hiCD11c+ DC precursors
expressed high levels of myeloid antigens CD11b and c-fms, but
low to moderate levels of Ia and CD86, implying that they are myeloid
precursor-derived cells. In response to GM-CSF and TNF-, they
differentiated into mature DCs with the phenotype of
CD11b/dullCD11c+ and obtained the expression
of high levels of CD40 and other markers of mature DC. As with their
precursors, these mature DCs continuously displayed abundant
c-fms mRNA and nonspecific esterase activity. Interestingly,
CD11b+hiCD11c+ DC precursors could
differentiate into macrophages in response to M-CSF, indicating that
CD11b+hiCD11c+ DC precursors may be an
intermediate-stage cell population of myeloid origin rather than being
restricted to DC commitment cells at the immature stage.
The observations by electron microscopy indicate that both
CD11b/dullCD11c+ and
CD11b+hiCD11c+ DC precursors contained
mitochondria and rough endoplasmic reticulum in the cytoplasm, while
their mature DC offspring had well-developed Golgi apparatus,
mitochondria, RER, and tubulovesicular system, particularly vesicles and multivesicular bodies near the Golgi apparatus. As previously described,27,33 the
tubulovesicular system and vesicles in DCs may be an intracellular
storage compartment of MHC class II molecules which has connections to
the lysosomal apparatus in the same region within the cells. For
successful expression of antigenic peptide-MHC class II complexes on
the cell surface, the endocytosed antigen needs to be in proximity to
newly synthesized class II MHC molecules in the specialized compartments of the endosome/lysosome system.34 Most
recently it has been shown that DCs can modulate these parameters to
control antigen presentation, and maturation of DC engages the
appropriate cellular component to stimulate the trafficking of MHC
class II molecules onto the cell surface.29,30,34 However,
several questions remain to be clarified: (1) the molecular basis of
the changes in MHC class II trafficking after forming the complexes with loaded antigens; (2) the signals responsible for constitutive membrane ruffling and endocytosis in immature DCs; and (3) the mechanisms for downregulating this response. All of these questions may
be critical for designing DC-based
immunotherapy.29,30,34,35 The existence of distinct
differentiation pathways mediated by CD11b/dullCD11c+ and
CD11b+hiCD11c+ DC precursors implies that
heterogeneous DCs might develop the capacity for driving MHC class II
and antigenic peptide complexes to the cell surface in a distinct
manner or at a distinct rate. Our findings may provide an important
tool for elucidating these questions by using purified and well-defined
differentiation or maturation stage of DC precursors based on
immunophenotypings; eg, the expression of CD11b and CD11c in our
system.
Based on the phenotype and differentiation potential, the DC precursor
subpopulations described here likely correspond to those generated from
human cord blood CD34+ HPCs as described by Caux et
al.14,15 The phenotype of
CD11b/dullCD11c+ precursor-derived DCs
appears identical to that of
CD14c-fmsCD1a+
precursor-derived DCs. CD11b+hiCD11c+
precursor-derived DC populations appear similar to
CD14+c-fms+CD1a
precursor-derived DCs.14,15 Since it has been
shown that
CD14c-fmsCD1a+ and
CD14+c-fms+CD1a
precursor cell-derived mature DCs play in vitro differential roles in
regulating cellular and humoral immune responses,
respectively,14,15 our findings might be helpful for
elucidating their individual biological function in vivo by using
various murine models.
Furthermore, it remains unclear whether the DC populations described
here correspond to those reported by Maraskovsky et al16 and Pulendran et al,17 who characterized several in vivo DC subsets including CD11bCD11c+,
CD11bdullCD11c+, and
CD11b+hiCD11c+ cell populations in the spleen,
but only CD11bCD11c+ DC subset in the
thymus, of Flt3L-treated mice.16,17 It is noted that in
Flt3L-treated mice CD11b+hiCD11c+-derived DCs,
which have been considered to be of myeloid precursor origin, cannot be
induced to differentiate into CD11bCD11c+
and CD11bdullCD11c+ cells in vitro by overnight
culture.17 CD11b+hiCD11c+ DC
precursors in our cultures can consistently not differentiate into the
same mature DCs with CD11b/dullCD11c+ DC
precursor-derived mature DCs in the expression of E-cadherin, c-fms, and nonspecific esterase activity; this
substantially supports the findings observed by Maraskovsky et al
and Pulendran et al that various DC subsets may develop in vivo along
distinct differentiation pathways.16,17 However, the DC and
DC precursor subsets described here were generated in the culture
system in vitro, which are apparently different from the DC
subpopulations in Flt3L-treated mice in vivo.16,17 It will
be difficult to directly compare at this time the DC and DC precursor
subsets generated in vitro with those DC subsets developed in vivo in
Flt3L-treated mice. Moreover, previous investigations have shown that
CD4low thymic precursor cells can differentiate in vivo
into CD8+ lymphoid mature DCs in mice. In contrast,
CD8 antigen cannot be detected on mature DCs derived in vitro from
the same CD4low thymic precursor cells stimulated with
various combinations of cytokines,36,37 indicating that
some other unknown factor might control DC differentiation and its
phenotype in vivo. It will be necessary to directly show the
differentiation pathways of DC subsets from
Linc-kit+ HPCs in vivo by taking
the advantage of animal models and/or by establishing in vitro
a culture system for generating lymphoid mature DCs, which may help to
elucidate the relationship of DCs and their precursor subsets generated
in vitro and those in vivo.
Several lines of evidence indicate that murine DCs bearing CD8
antigen are of lymphoid-precursor origin, whereas DCs expressing high
levels of CD11b antigen are derived from
myeloid-precursors.3-6,36,37 Furthermore, early murine
thymic precursors can only differentiate into lymphoid cells and DCs,
but not myeloid cells,3,36,37 suggesting that the
topographic organization of heterogeneous DC may have been already
determined, at least in part, at the progenitor
levels.14,38-46 Therefore, further characterization of the
phenotype of murine DC precursor or DC-committed progenitor cells,
which account for the generation of
CD11b/dullCD11c+ and
CD11b+hiCD11c+ precursors, respectively, will
prove to be valuable for investigating the cellular and molecular
mechanisms of DC differentiation from HPCs in vivo and in vitro.
| |
FOOTNOTES |
Submitted December 1, 1997;
accepted March 5, 1998.
Supported in part by Grants-in-Aid from the Ministry of Education,
Culture, Science, and Sports of the Japanese Government.
Address reprint requests to Kouji Matsushima, MD, PhD, Department of
Molecular Preventive Medicine, School of Medicine, The University of
Tokyo, 7-3-1, Hongo, Bunkyoku, Tokyo 113, Japan; e-mail:
koujim{at}m.u-tokyo.ac.jp.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We express our sincere gratitude to Dr R.M. Steinman (The Rockefeller
University, New York, NY) for his kind gift of MoAbs to DEC-205
(NLDC145) and 33D1; and to Dr T. Sudo (Basic Research Institute of
Toray Co, Kanagawa, Japan) for his generous gift of anti-c-kit
MoAb, GM-CSF, and SCF. We also highly appreciate Dr J.J. Oppenheim
(NCI-FCRDC, Frederick, MD) for his critical review of the manuscript.
| |
REFERENCES |
1.
Steinman RM,
Kaplan G,
Witmer MD,
Cohn ZA:
Identification of a novel cell type in peripheral lymphoid organs of mice. V. Purification of spleen dendritic cells, new surface markers, and maintenance in vitro.
J Exp Med
149:1,
1979[Abstract/Free Full Text]
2.
Steimnman RM:
The dendritic cell system and its role in immunogenicity.
Annu Rev Immunol
9:271,
1991[Medline]
[Order article via Infotrieve]
3.
Ardavin C:
Thymic dendritic cells.
Immunol Today
18:350,
1997[Medline]
[Order article via Infotrieve]
4.
Vremec DV,
Zorbas M,
Scolly R,
Saunders DJ,
Ardavin CF,
Wu L,
Shortman K:
The surface phenotype of dendritic cells purified from mouse thymus and spleen: Investigation of the CD8 expression by a subpopulation of dendritic cells.
J Exp Med
176:47,
1992[Abstract/Free Full Text]
5.
Shortman K,
Wu L,
Ardavin C,
Vremec D,
Stozik F,
Winkel F,
Suss G:
Thymic dendritic cells: surface phenotype, developmental origin and function.
Adv Exp Med Biol
378:21,
1995[Medline]
[Order article via Infotrieve]
6.
Wu L,
Vremec D,
Ardavin C,
Winkel K,
Suss G,
Georgiou H,
Cook W,
Shortman K:
Mouse thymus dendritic cells: Kinetics of development and changes in surface markers during maturation.
Eur J Immunol
25:418,
1995[Medline]
[Order article via Infotrieve]
7.
Zhang Y,
Mukaida N,
Wang JB,
Harada A,
Akiyama M,
Matsushima K:
Induction of dendritic cell differentiation by granulocyte-macrophage colony-stimulating factor, stem cell factor, and tumor necrosis factor in vitro from lineage phenotypes negative c-kit+ murine hematopoietic progenitor cells.
Blood
90:4842,
1997[Abstract/Free Full Text]
8.
Lentz A,
Heine M,
Schuler G,
Romani N:
Human and murine dermis contain dendritic cells.
J Clin Invest
92:2587,
1993
9.
Nestle FO,
Zheng XG,
Thompson CB,
Turka LA,
Nickoloff BJ:
Characterization of dermal dendritic cells obtained from normal human skin reveals phenotypic and functional distinctive subsets.
J Immunol
151:6535,
1993[Abstract]
10.
Doherty U,
Peng M,
Gezelter S,
Swiggard WJ,
Betjes M,
Bhardwaj N,
Steinman RM:
Human blood contains two subsets of dendritic cells, one immunologically mature and the other immature.
Immunology
82:487,
1994[Medline]
[Order article via Infotrieve]
11.
Weissman D,
Li Y,
Anaworanich J,
Zhaou LJ,
Adelberger J,
Tedder TF,
Basler M,
Fauci AS:
Three populations of cells with dendritic morphology exist in peripheral blood, only one of which is infectable with human immunodeficiency virus type 1.
Proc Natl Acad Sci USA
92:826,
1995[Abstract/Free Full Text]
12.
Ardavin C,
Wu L,
Ferrero I,
Shortman K:
Mouse thymic dendritic cell subpopulations.
Immunol Lett
38:19,
1993[Medline]
[Order article via Infotrieve]
13.
Suss G,
Shortman K:
A subclass of dendritic cell kills CD4 T cells via Fas/Fas-ligand-induced apoptosis.
J Exp Med
183:1789,
1996[Abstract/Free Full Text]
14.
Caux C,
Vanbervliet B,
Massacrier C,
Denzutter-Dambuyant C,
Saint-Vis Blandine,
Jacquet C,
Yoneda K,
Imamura S,
Schmitt D,
Banchereau J:
CD34+ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to GM-CSF + TNF.
J Exp Med
184:695,
1996[Abstract/Free Full Text]
15.
Caux C,
Massachrier C,
Vanbervliet B,
Dubois B,
Durand I,
Cella M,
Lanzavecchia,
Banchereau J:
CD34+ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to granulocyte-macrophage colony stimulating factor plus tumor necrosis factor : II. Functional analysis.
Blood
90:1458,
1997[Abstract/Free Full Text]
16.
Maraskovsky E,
Brasel K,
Teepe M,
Roux ER,
Lyman SD,
Shortman K,
McKenna HJ:
Dramatic increase in the numbers of functionally mature dendritic cells in Flt3 ligand-treated mice: Multiple dendritic cell subpopulations identified.
J Exp Med
184:1953,
1996[Abstract/Free Full Text]
17.
Pulendran B,
Lingappa J,
Kennedy MK,
Smith J,
Teepe M,
Rudensky A,
Maliszewski CR,
Maraskovsky E:
Developmental pathways of dendritic cells in vivo: Distinct function, phenotype, and localization of dendritic cell subsets in Flt-3 ligand-treated mice.
J Immunol
159:2222,
1997[Abstract/Free Full Text]
18.
Goeddel DV,
Aggaewal BB,
Gray PW,
Leung DW,
Nedwin GE,
Paladino MA,
Patton JS,
Pennica D,
Ahepard HM,
Sugarman BJ,
Wong GHW:
Tumor necrosis factors: Gene structure and biological activities.
Cold Spring Harb Symp Quant Biol
1:597,
1986
19.
Nishikawa S,
Ogawa M,
Kusakabe M,
Kunisda T,
Era T,
Sakakura T,
Nishikawa SI:
In utero manipulation of coat color formation by monoclonal anti-c-kit antibody: Two distinct waves of c-kit dependency during melanocyte development.
EMBO J
10:2111,
1991[Medline]
[Order article via Infotrieve]
20.
Kraal G,
Breel M,
Jaanse M,
Bruin G:
Langerhans cells, veiled cells, and interdigitating cells in the mouse recognized by a monoclonal antibody.
J Exp Med
163:981,
1986[Abstract/Free Full Text]
21.
Swiggard WJ,
Mirza A,
Nussenzweig MC,
Steinman RM:
DEC-205, a 250-kD protein abundant on mouse dendritic cells and thymic epithelium that is detected by the monoclonal antibody NLDC-145: Purification, characterization and N-terminal amino acid sequence.
Cell Immunol
165:302,
1995[Medline]
[Order article via Infotrieve]
22.
Zhang Y,
Harada A,
Bluethmann H,
Wang JB,
Nakao S,
Mukaida N,
Matsushima K:
Tumor necrosis factor (TNF) is a physiological regulator of hematopoietic progenitor cells: Increase of early hematopoietic progenitor cells in TNF receptor p55-deficient mice in vivo and potent inhibition of progenitor cell proliferation by TNF in vitro.
Blood
86:2930,
1995[Abstract/Free Full Text]
23.
Wang JB,
Zhang Y,
Kasahara T,
Harda A,
Matsushima K,
Mukaida N:
Detection of mouse IL-8 receptor homologue expression on peripheral blood leukocytes and mature myeloid lineage cells in bone marrow.
J Leukoc Biol
60:372,
1996[Abstract]
24.
Rothwell VM,
Rohrschneider LR:
Murine c-fms cDNA: cloning, sequence analysis and retroviral expression.
Oncogene Res
1:311,
1987[Medline]
[Order article via Infotrieve]
25.
Winzler C,
Rovere P,
Rescigno M,
Granucci F,
Penna G,
Adorini L,
Zimmermann VS,
Davoust J,
Castagnoli PR:
Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures.
J Exp Med
185:317,
1997[Abstract/Free Full Text]
26.
Cohen PJ,
Cohen PA,
Rosenberg SA,
Katz SI,
Mule JJ:
Murine epidermal Langerhans cells and splenic dendritic cells present tumor-associated antigens to primed T cells.
Eur J Immunol
24:315,
1994[Medline]
[Order article via Infotrieve]
27.
Umezu H,
Naito M,
Inaba K,
Takahashi K:
Ultrastructural and immunophenotypic differentiation of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor (GM-CSF).
J Submicrosc Cytol Pathol
27:227,
1995[Medline]
[Order article via Infotrieve]
28.
Borkowski T,
Van Dyke BJ,
Schwarzenberger K,
McFarland VW,
Farr AG,
Udey MC:
Expression of E-cadherin by murine dendritic cell: E-cadherin as a dendritic differentiation antigen characteristic of epidermal Langerhans cells and related cells.
Eur J Immunol
24:2767,
1994[Medline]
[Order article via Infotrieve]
29.
Cella M,
Engering A,
Pinet V,
Pieters J,
Lanzavecchia A:
Inflammatory stimuli induce accumulation of MHC class II complexes on dendritic cells.
Nature
388:782,
1997[Medline]
[Order article via Infotrieve]
30.
Pierre P,
Turley SJ,
Gatti E,
Hull M,
Meltzer J,
Mirza A,
Inaba K,
Steinman RM,
Mellman I:
Developmental regulation of MHC class II transport in mouse dendritic cells.
Nature
388:787,
1997[Medline]
[Order article via Infotrieve]
31.
Inaba K,
Inaba M,
Deguchi K,
Hagi R,
Yasumizu S,
Ikehara S,
Muramatsu S,
Steinman RM:
Granulocytes, macrophages, and dendritic cells arise from a common major histocompatibility complex class II-negative progenitor in mouse bone marrow.
Proc Natl Acad Sci USA
90:3038,
1993[Abstract/Free Full Text]
32.
Inaba K,
Inaba M,
Romani N,
Aya H,
Deguchi M,
Ikehara S,
Muranatsu S,
Stieinman RM:
Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony stimulating factor.
J Exp Med
176:1693,
1992[Abstract/Free Full Text]
33.
Hoefstmit ECM:
Immunohistochemical characterization of dendritic cells
, in Kamperdijk EWA,
Nieuwenhuis P,
Hoefsmit EC
(eds):
Dendritic Cells in Fundamentals and Clinical Immunology.
New York, NY, Plenum
, 1993
, p 1
34.
Watts C:
Inside the gearbox of the dendritic cell.
Nature
388:724,
1997[Medline]
[Order article via Infotrieve]
35.
Steinman RM:
Dendritic cells and immune-based therapies.
Exp Hematol
24:859,
1996[Medline]
[Order article via Infotrieve]
36.
Saunders D,
Lucas K,
Ismaili J,
Wu L,
Maraskovsky E,
Dunn A,
Shortman K:
Dendritic cell development in culture from thymic precursor cells in the absence of granulocyte/macrophage colony stimulating factor.
J Exp Med
184:2185,
1996[Abstract/Free Full Text]
37.
Wu L,
Li CL,
Shortman K:
Thymic dendritic cell precursors: relationship to the T lymphocyte lineage and phenotype of the dendritic cell progeny.
J Exp Med
184:903,
1996[Abstract/Free Full Text]
38.
Young JM,
Szabolcs P,
Moor MAS:
Identification of dendritic cell colony-forming units among normal human CD34+ bone marrow progenitors that expanded by c-kit-ligand and yield pure dendritic cell colonies in the presence of granulocyte/macrophage colony-stimulating factor and tumor necrosis factor .
J Exp Med
182:1111,
1995[Abstract/Free Full Text]
39.
Rosenzwajg M,
Canque B,
Gluckman JC:
Human dendritic cell differentiation pathway from CD34+ hematopoietic precursor cells.
Blood
87:535,
1996[Abstract/Free Full Text]
40.
Strunk D,
Rappersberger K,
Egger C,
Strobl H,
Kromer E,
Elbe A,
Maurer D,
Stingl G:
Generation of human dendritic cell/Langerhans cells from circulating CD34+ hematopoietic progenitor cells.
Blood
87:1292,
1996[Abstract/Free Full Text]
41.
Akagawa KS,
Takasuka N,
Nozaki Y,
Komuro I,
Azuma M,
Ueda M,
Naito M,
Takahashi K:
Generation of CD1a+RelB+ dendritic cells and tartrate-resistant acid phosphatase-positive osteoclast-likt multinucleated giant cells from human monocytes.
Blood
88:4029,
1996[Abstract/Free Full Text]
42.
Borkwski TA,
Letterio JJ,
Farr AG,
Udey MC:
A role for endogenous transforming growth factor 1 in Langerhans cell biology: The skin of transforming growth factor 1 null mice is devoid of epidermal Langerhans cell.
J Exp Med
184:2417,
1996[Abstract/Free Full Text]
43.
Burkly L,
Hession C,
Ogata L,
Eilly C,
Marconi LA,
Olson D,
Tizard R,
Cate R,
Lo D:
Expression of relB is required for the development of thymic medulla and dendritic cells.
Nature
373:531,
1995[Medline]
[Order article via Infotrieve]
44.
Weih F,
Carraso D,
Durham SK,
Barton DS,
Rizzo CA,
Ryseck RP,
Lira SA,
Bravo R:
Multiorgan inflammation and hematopoietic abnormalities in mice with a targeted disruption of relB, a member of the NF-kB/Rel family.
Cell
80:331,
1995[Medline]
[Order article via Infotrieve]
45.
Galy A,
Travis M,
Cen D,
Chen B:
Human T, B, natural killer, and dendritic cells arise from a common bone marrow progenitor cell subset.
Immunity
3:459,
1995[Medline]
[Order article via Infotrieve]
46.
Strunk D,
Egger C,
Leitner G,
Hanau D,
Stingl G:
A skin homing molecule defines the Langerhans cells progenitor in human peripheral blood.
J Exp Med
185:1131,
1997[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
H.-J. Ko, J.-M. Lee, Y.-J. Kim, Y.-S. Kim, K.-A Lee, and C.-Y. Kang
Immunosuppressive Myeloid-Derived Suppressor Cells Can Be Converted into Immunogenic APCs with the Help of Activated NKT Cells: An Alternative Cell-Based Antitumor Vaccine
J. Immunol.,
February 15, 2009;
182(4):
1818 - 1828.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. He, Q. Cao, H. Yoneyama, H. Ge, Y. Zhang, and Y. Zhang
MIP-3{alpha} and MIP-1{alpha} rapidly mobilize dendritic cell precursors into the peripheral blood
J. Leukoc. Biol.,
December 1, 2008;
84(6):
1549 - 1556.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. He, Q. Cao, Y. Qiu, J. Mi, J. Z. Zhang, M. Jin, H. Ge, S. G. Emerson, Y. Zhang, and Y. Zhang
A New Approach to the Blocking of Alloreactive T Cell-Mediated Graft-versus-Host Disease by In Vivo Administration of Anti-CXCR3 Neutralizing Antibody
J. Immunol.,
December 1, 2008;
181(11):
7581 - 7592.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Zhang, E. Hexner, D. Frank, and S. G. Emerson
CD4+ T Cells Generated De Novo from Donor Hemopoietic Stem Cells Mediate the Evolution from Acute to Chronic Graft-versus-Host Disease
J. Immunol.,
September 1, 2007;
179(5):
3305 - 3314.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Mao, V. Paharkova-Vatchkova, J. Hardy, M. M. Miller, and S. Kovats
Estrogen Selectively Promotes the Differentiation of Dendritic Cells with Characteristics of Langerhans Cells
J. Immunol.,
October 15, 2005;
175(8):
5146 - 5151.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
O. M. Z. Howard, H. F. Dong, S. B. Su, R. R. Caspi, X. Chen, P. Plotz, and J. J. Oppenheim
Autoantigens signal through chemokine receptors: uveitis antigens induce CXCR3- and CXCR5-expressing lymphocytes and immature dendritic cells to migrate
Blood,
June 1, 2005;
105(11):
4207 - 4214.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Menges, T. Baumeister, S. Rossner, P. Stoitzner, N. Romani, A. Gessner, and M. B. Lutz
IL-4 supports the generation of a dendritic cell subset from murine bone marrow with altered endocytosis capacity
J. Leukoc. Biol.,
April 1, 2005;
77(4):
535 - 543.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Zhang, G. Joe, E. Hexner, J. Zhu, and S. G. Emerson
Alloreactive Memory T Cells Are Responsible for the Persistence of Graft-versus-Host Disease
J. Immunol.,
March 1, 2005;
174(5):
3051 - 3058.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Kissenpfennig, S. Ait-Yahia, V. Clair-Moninot, H. Stossel, E. Badell, Y. Bordat, J. L. Pooley, T. Lang, E. Prina, I. Coste, et al.
Disruption of the langerin/CD207 Gene Abolishes Birbeck Granules without a Marked Loss of Langerhans Cell Function
Mol. Cell. Biol.,
January 1, 2005;
25(1):
88 - 99.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. de Repentigny, D. Lewandowski, and P. Jolicoeur
Immunopathogenesis of Oropharyngeal Candidiasis in Human Immunodeficiency Virus Infection
Clin. Microbiol. Rev.,
October 1, 2004;
17(4):
729 - 759.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Zhang, G. Joe, J. Zhu, R. Carroll, B. Levine, E. Hexner, C. June, and S. G. Emerson
Dendritic cell-activated CD44hiCD8+ T cells are defective in mediating acute graft-versus-host disease but retain graft-versus-leukemia activity
Blood,
May 15, 2004;
103(10):
3970 - 3978.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. C. O'Neill and H. L. Wilson
Limitations with in vitro production of dendritic cells using cytokines
J. Leukoc. Biol.,
April 1, 2004;
75(4):
600 - 603.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Zhang, H. Yoneyama, Y. Wang, S. Ishikawa, S.-i. Hashimoto, J.-L. Gao, P. Murphy, and K. Matsushima
Mobilization of Dendritic Cell Precursors Into the Circulation by Administration of MIP-1{alpha} in Mice
J Natl Cancer Inst,
February 4, 2004;
96(3):
201 - 209.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Hamrah, L. Chen, Q. Zhang, and M. R. Dana
Novel Expression of Vascular Endothelial Growth Factor Receptor (VEGFR)-3 and VEGF-C on Corneal Dendritic Cells
Am. J. Pathol.,
July 1, 2003;
163(1):
57 - 68.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Hamrah, Y. Liu, Q. Zhang, and M. R. Dana
The Corneal Stroma Is Endowed with a Significant Number of Resident Dendritic Cells
Invest. Ophthalmol. Vis. Sci.,
February 1, 2003;
44(2):
581 - 589.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Zhang, W. D. Shlomchik, G. Joe, J.-P. Louboutin, J. Zhu, A. Rivera, D. Giannola, and S. G. Emerson
APCs in the Liver and Spleen Recruit Activated Allogeneic CD8+ T Cells to Elicit Hepatic Graft-Versus-Host Disease
J. Immunol.,
December 15, 2002;
169(12):
7111 - 7118.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. A. Hume, I. L. Ross, S. R. Himes, R. T. Sasmono, C. A. Wells, and T. Ravasi
The mononuclear phagocyte system revisited
J. Leukoc. Biol.,
October 1, 2002;
72(4):
621 - 627.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Wang, Y. Zhang, H. Yoneyama, N. Onai, T. Sato, and K. Matsushima
Identification of CD8alpha +CD11c- lineage phenotype-negative cells in the spleen as committed precursor of CD8alpha + dendritic cells
Blood,
June 28, 2002;
100(2):
569 - 577.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Miyamoto, O. Ohneda, F. Arai, K. Iwamoto, S. Okada, K. Takagi, D. M. Anderson, and T. Suda
Bifurcation of osteoclasts and dendritic cells from common progenitors
Blood,
October 15, 2001;
98(8):
2544 - 2554.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Canque, S. Camus, A. Dalloul, E. Kahn, M. Yagello, C. Dezutter-Dambuyant, D. Schmitt, C. Schmitt, and J. C. Gluckman
Characterization of dendritic cell differentiation pathways from cord blood CD34+CD7+CD45RA+ hematopoietic progenitor cells
Blood,
December 1, 2000;
96(12):
3748 - 3756.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Zhang, N. Chirmule, G.-p. Gao, and J. Wilson
CD40 Ligand-Dependent Activation of Cytotoxic T Lymphocytes by Adeno-Associated Virus Vectors In Vivo: Role of Immature Dendritic Cells
J. Virol.,
September 1, 2000;
74(17):
8003 - 8010.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
Y. Zhang, Y. Zhang, Y. Wang, M. Ogata, S.-i. Hashimoto, N. Onai, and K. Matsushima
Development of dendritic cells in vitro from murine fetal liver-derived lineage phenotype-negative c-kit+ hematopoietic progenitor cells
Blood,
January 1, 2000;
95(1):
138 - 146.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Ogata, Y. Zhang, Y. Wang, M. Itakura, Y.-y. Zhang, A. Harada, S.-i. Hashimoto, and K. Matsushima
Chemotactic Response Toward Chemokines and Its Regulation by Transforming Growth Factor-{beta}1 of Murine Bone Marrow Hematopoietic Progenitor Cell-Derived Different Subset of Dendritic Cells
Blood,
May 15, 1999;
93(10):
3225 - 3232.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Zhang, Y.-y. Zhang, M. Ogata, P. Chen, A. Harada, S.-i. Hashimoto, and K. Matsushima
Transforming Growth Factor-beta 1 Polarizes Murine Hematopoietic Progenitor Cells to Generate Langerhans Cell-Like Dendritic Cells Through a Monocyte/Macrophage Differentiation Pathway
Blood,
February 15, 1999;
93(4):
1208 - 1220.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract