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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 4 (February 15), 1999:
pp. 1208-1220
Transforming Growth Factor- 1 Polarizes Murine Hematopoietic
Progenitor Cells to Generate Langerhans Cell-Like Dendritic Cells
Through a Monocyte/Macrophage Differentiation Pathway
By
Yi Zhang,
Yan-yun Zhang,
Masafumi Ogata,
Pan Chen,
Akihisa Harada,
Shin-ichi Hashimoto, and
Kouji Matsushima
From the Department of Molecular Preventive Medicine and CREST,
School of Medicine, The University of Tokyo, Tokyo, Japan.
 |
ABSTRACT |
We have recently demonstrated that
CD11b /dullCD11c+ and
CD11b+hiCD11c+ dendritic cell (DC)
precursor subsets represent two distinct DC differentiation pathways
from murine bone marrow lineage-phenotype negative
(Lin)c-kit+ hematopoietic progenitor cells
(HPCs) stimulated with granulocyte-macrophage colony-stimulating factor
(GM-CSF) + stem cell factor (SCF) + tumor necrosis factor 
(TNF). We show here that transforming growth factor- 1 (TGF-1)
significantly inhibits the generation of these
CD11b/dullCD11c+ and
CD11b+hiCD11c+ DC precursors.
Phenotypically, this inhibitory effect was accompanied by markedly
suppressed expression of Ia and CD86 antigens as well as major
histocompatibility complex (MHC) class II transactivator (CIITA) and
CC-chemokine receptor 7 (CCR7) mRNAs in
Linc-kit+ HPC cultures stimulated with
GM-CSF + SCF + TNF at day 6. TGF-1 could also suppress mature
DC differentiation from CD11b+hiCD11c+ DC
precursors, but not the differentiation from
CD11b/dullCD11c+ DC precursors. In the
absence of TNF, TGF-1 markedly suppressed the expression of CIITA
and CCR7 mRNAs in GM-CSF + SCF-stimulated Linc-kit+ HPCs at either day 6 or day 12 and induced the differentiation solely into monocytes/macrophages as
evident in morphology, active phagocytic, and endocytic activities.
These cells expressed high levels of F4/80 and E-cadherin antigens, but
low or undetectable levels of Ia, CD86, and CD40 molecules. However,
upon the stimulation with TNF + GM-CSF, these cells could further
differentiate into mature DCs expressing high levels of Ia and
E-cadherin, characteristics for Langerhans cells (LCs), and gained the
capacity of enhancing allogenic MLR. Taken together, all of these
findings suggest that TGF-1 polarizes murine HPCs to generate
LC-like DCs through a monocyte/macrophage differentiation pathway.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
DENDRITIC CELL (DC) development from
hematopoietic progenitor cells (HPCs) has been classified into four
stages: proliferating DC progenitors, nonproliferating DC precursors,
immature antigen capturing DCs, and mature DCs with T-cell stimulatory
function.1 Accordingly, heterogeneous DC subpopulations in
different tissues may originate from distinct DC progenitors/precursors
and/or from the same progenitors/precursors induced by
differential sets of cytokines in situ.1-9 Several
cytokines have been shown to regulate the growth, differentiation, and
survival of DCs.4-16 Both stem cell factor
(SCF) and Flt3 ligand sustain the growth of DC
progenitors.4-7,14 Administration of Flt3 ligand
stimulates outgrowth of at least three DC subsets, such as
CD11bCD11c+,
CD11bdullCD11c+, and
CD11b+hiCD11c+ DC subsets in
mice.6,7 Granulocyte-macrophage colony-stimulating factor
(GM-CSF) and interleukin-3 (IL-3) enhance DC differentiation into
intermediate stage, whereas tumor necrosis factor  (TNF) and CD40
ligand (CD40L) stimulate final maturation of DCs.4,5,9-16 However, mature DCs in vivo are small leukocyte
populations.1,2 It remains unclear how growth and
differentiation of DC progenitor or precursor cells are regulated and
which cytokines may account for the generation of heterogeneous DC
subpopulations with different tissue distributions and functions.
Transforming growth factor-1 (TGF-1) is a pleiotropic cytokine
produced by many types of cells.17,18 Accumulating evidence indicates that TGF-1 plays an essential role for the generation of
Langerhans cells (LCs) in vivo and in vitro.10,19,20 LCs are DCs that exist in the epidermis.21 Disruption of the
TGF-1 gene results in a profoundly developmental deficiency of LCs
in mice.20 Furthermore, in concert with GM-CSF and IL-4,
TGF-1 promotes LC differentiation from human peripheral blood
monocytes,10 suggesting that TGF-1 is an essential
cytokine for LC differentiation. However, the cellular and molecular
mechanisms for TGF-1 to regulate LC differentiation from early HPCs
remain to be elucidated. It has been demonstrated that TGF-1
requires collaboration with GM-CSF and TNF to induce DC
differentiation from HPCs,19,22 whereas, in the absence of
TNF, TGF-1 completely suppressed mature DC generation from
GM-CSF-induced murine bone marrow progenitor cells.22
Moreover, targeted disruption of TGF-1 gene in mice shows a
prominent feature with enhanced expression of major histocompatibility complex (MHC) class II molecules, which results in the progressive multifocal inflammatory processes and autoimmune
diseases.23,24 These data suggest that TGF-1 may
function as a natural suppressor of the inflammatory process through
regulating the expression and function of MHC class II
antigen.23,24 Because MHC class II antigen is highly
expressed in DCs that play a central role in controlling immune
responses,1,2 these observations suggest that the bipotent
role of TGF-1 in regulating the generation of DCs is dependent on
the differentiation state of HPCs, DC precursors, and the supplemented cytokines.
We have recently demonstrated in vitro that
CD11b/dullCD11c+ and
CD11b+hiCD11c+ DC precursors are two distinct
nonproliferating DC precursors generated from murine
Linc-kit+ HPCs in the presence of GM-CSF + SCF + TNF.9 Using this DC differentiation
model,9,25 we investigated here the effect of TGF-1 on
DC differentiation from murine Linc-kit+
HPCs. We describe that TGF-1 could markedly inhibit the generation of CD11b/dullCD11c+ and
CD11b+hiCD11c+ DC precursor subsets by
polarizing HPC differentiation into monocytes/macrophages with the
capacity to differentiate into LC-like DCs in vitro.
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MATERIALS AND METHODS |
Cytokines and antibodies.
Recombinant murine SCF and GM-CSF were kindly provided by Dr T. Sudo
(Basic Research Institute of Toray Co, Kanagawa, Japan) and by Kirin
Brewery Co (Tokyo, Japan). Human TGF-1 was purchased from R&D System
(Minneapolis, MN). Mouse TNF was produced as described
previously.26 These cytokines were used at the optimal concentrations as previously described.9,25 An anti-c-kit antibody (ACK-2) was kindly provided by Dr T. Sudo and conjugated with
biotin by using a NHS-Biotin kit (Pharmacia-Biotech, Uppsala, Sweden)
according to the manufacturer's instructions.27 A rat monoclonal antibody (MoAb) to murine DCs, DEC-205 (NLDC145), was a
generous gift of Dr R.M. Steinman (Rockefeller University, New York,
NY).28,29 The MoAb to mouse E-cadherin was purchased from
Dainipon Pharmaceutical Co (Osaka, 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 Clea 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.
Bone marrow cells were obtained by aspirating femurs and tibiae of 8- to 10-week-old female mice. Linc-kit+
HPCs were isolated from nonadherent bone marrow mononuclear cells (MNCs) using an EPICS ELITE cell sorter (Coulter Electronics, Hialeah,
FL) as previously described.9,25 In brief, nonadherent MNCs
were stained with an indirect staining composed of a 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 described at a cell concentration of 1 to 3 × 104 cells/mL in Iscove's modified Dulbecco 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.10,26 TGF-1 was added in the cultures in a
various combination as indicated. Optimal conditions were maintained by
splitting these cultures at day 4 and exchanged the medium containing
fresh cytokines every 3 to 4 days.
In some experiments, CD11b-/dullCD11c+ and
CD11b+hiCD11c+ DC precursor subsets were sorted
at day 6 from Linc-kit+ HPC cultures
stimulated with GM-CSF, SCF, and TNF as previously described.9 In some experiments, TGF-1-induced
Linc-kit+ HPC cultures stimulated with
GM-CSF + SCF were collected at day 12, washed twice, and recultured in
the presence of GM-CSF + TNF for an additional 3 to 5 days. All of
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 greater than 98%.
Immunofluorescence analysis.
Immunofluorescence analyses were performed as previously
described.9,25 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 (CyC)-labeled streptavidin and PE-conjugated goat antirat
IgG(Fab')2 antibodies. The cells were then stained
with FITC-conjugated various MoAbs. In some experiments, the cells were
first stained with a rat anti-E-cadherin MoAb, followed by staining
with FITC-conjugated goat antirat IgG(Fab')2
antibodies and a PE-labeled anti-Ia MoAb. In other experiments, the
cells were first stained with biotinylated antibodies and shown by
CyC-conjugated streptavidin, followed by staining with PE-labeled
anti-CD11c and FITC-conjugated anti-CD11b antibodies. 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 2 × 105 indicated
cells using RNAzol B (Biotex Laboratories Inc, Houston, TX) according
to the manufacturer's instructions. First-strand cDNA was synthesized from 200 ng of total RNAs in 25-µL reaction volume using an RT-PCR kit (Takara Shuzo, Kyoto, Japan) with random primers. Thereafter, cDNA
was amplified for 30 cycles consisting of 94°C for 30 seconds, 57°C for 1 minute, and 72°C for 1.5 minutes with the CIITA
oligonucleotide primers (5'-CCAGAACTGGTTGTAGAGCC-3' and
5'-CAGCTTGCTAGGCTCCAATT-3'), which specifically result in a
500-bp cDNA encoding CIITA gene.30,31 CCR7 mRNA was
examined by using oligonucleotide primers
(5'-CATCAGCATTGACCGCTACGT-3' and
5'-GGTACGGATGATAATGAGGTAGCA-3'), which are specific for
murine CCR7.32 As a control, mouse -actin transcript was
amplified in parallel as previously described.9,25 The PCR
products were fractionated on 1.5% agarose gel or 5% polyacrylamid
gel and visualized by ethidium bromide staining.
Endocytosis and phagocytosis.
The endocytosis experiments were performed as previously
reported.33 In brief, the 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. The
phagocytosis experiments were performed as reported.34 Briefly, the cells were harvested from the cultures and resuspended at
approximately 4 to 5 × 105/mL in complete IMDM. Eight
microliters of FITC-latex beads of 0.75 µm diameters (Carboxylate
Microspheres, Wako, Japan) were added to the cell suspension and mixed
well. The cells were incubated for 0 or 30 minutes at 37°C. The
incubation was extinguished by adding ice-cold 5% BSA-0.02% azide-PBS
and washed three times with 2.5% fetal calf serum (FCS)-0.02%
azide-PBS. To confirm the endocytic and phagocytic activity of DC
precursors, these cells were stained again with a rat-antimouse CD11b
MoAb and a biotinylated hamster antimouse CD11c MoAb, followed by
staining with PE-conjugated goat antirat IgG(Fab')2
antibodies and CyC-conjugated streptavidin. Finally, the percentage and
density of FITC-positive cells were examined based on tri-color
analysis by gating on the
CD11b/dullCD11c+/dull cell population
using a cell sorter as described.9
MLR.
Splenic MNCs were prepared as described previously from allogenic mice
(Balb/c).9 The adherent cells were first removed by
incubating them at 37°C for 60 minutes in IMDM medium containing 10% 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 antirat IgG and antimouse 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),35 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). 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 using a microplate immunoreader.
Nonspecific esterase (NSE) staining.
Cells were cytocentrifuged for 5 minutes at 500 rpm on a microscope
slide and used for NSE staining (-naphthyl acetate esterase staining
kit; Sigma) according the instructions of the manufacturer.
Statistical analysis.
Differences were evaluated using the Student's t-test.
P values of less than .05 were considered to be statistically significant.
| |
RESULTS |
TGF-1 inhibits the generation of
CD11b/dullCD11c+ and
CD11b+hiCD11c+ DC precursors from
Linc-kit+ HPCs at day 6.
To elucidate the role of TGF-1 in DC generation,
Linc-kit+ HPCs were stimulated with
GM-CSF + SCF + TNF in the presence of various doses of TGF-1
ranging from 0 to 2.5 ng/mL, as indicated. Cells were then first
analyzed at day 6, when two distinct
CD11b/dullCD11c+ (10% ± 3.5%) and
CD11b+hiCD11c+ (26% ± 5.6%) DC precursor
subsets could be clearly identified. Addition of TGF-1
dose-dependently decreased the generation of CD11b/dullCD11c+ and
CD11b+hiCD11c+ DC precursor subsets to 2.0% ± 1.5% and 5% ± 3.5%, respectively (Fig 1A). Moreover, the absolute numbers of
the two DC precursor cells were also markedly reduced more than
fourfold and threefold, respectively (Fig 1B). The maximal inhibitory
effect of TGF-1 was reached at a concentration of 2.5 ng/mL;
therefore, this dose was used in the following experiments, unless
otherwise indicated. In contrast, TGF-1 markedly increased the
CD11b+hiCD11c cell fraction in GM-CSF + SCF + TNF-stimulated Linc-kit+
cultures (Fig 2A and B) and most of these
CD11b+hiCD11c cells were Gr-1 negative
(data not shown), suggesting that TGF-1 might potentiate the
differentiation of HPCs into monocytes/macrophages. Interestingly, in
the absence of TNF, GM-CSF + SCF could induce the generation of
CD11b+hiCD11c+ DC precursors rather than
CD11b-/dullCD11c+ ones, which was completely
blocked by the addition of TGF-1 (Fig 2A and B).
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| Fig 1.
TGF-1 dose-dependently inhibited the generation of
CD11b/dullCD11c+ and
CD11b+hiCD11c+ DC precursors at day 6 from
murine Linc-kit+ HPCs stimulated with
GM-CSF + SCF + TNF. (A) The percentage of
CD11b/dullCD11c+ and
CD11b+hiCD11c+ DC precursors. (B) The
absolute numbers of CD11b/dull
CD11c+ and CD11b+hiCD11c+
DC precursors. The data represent the mean value ± SD of the
percentage and numbers of the two DC precursor subpopulations observed
in more than five experiments. *P < .05 significance as compared with those cultures in the absence of
TGF-1.
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| Fig 2.
TGF-1 enhanced differentiation of
CD11b+hiCD11c cells from murine
Linc-kit+ HPCs in the presence of GM-CSF + SCF with or without TNF at day 6. (A) Dose-dependent induction
of CD11b+hiCD11c cells by TGF-1 in the
cultures stimulated with GM-CSF + SCF + TNF. The data represent
the mean value ± SD of the percentage of
CD11b+hiCD11c cells observed in more than
five experiments. *P < .05 significance as compared with the
cultures in the absence of TGF-1. (B) Induction of
CD11b/dullCD11c+,
CD11b+hiCD11c+, and
CD11b+hiCD11c cells by TGF-1 in
Linc-kit+ HPC cultures stimulated by
various combination of cytokines as indicated. Quads were set up on the
isotype-matched control dot plot, and the results are representative of
more than five experiments.
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To negate the possibility that TGF-1 might simply downregulate the
expression of CD11c, we also examined at day 6 the expression of Ia and
CD86 antigens that have been demonstrated to be highly expressed on
CD11b/dullCD11c+ DC precursor cells, but
moderately on CD11b+hiCD11c+ DC precursor
cells. As shown in Fig 3A and B, TGF-1
significantly decreased the generation of
Ia+CD86+ cells in
Linc-kit+ HPC cultures stimulated with
GM-CSF + SCF + TNF, supporting the notion that TGF-1 could indeed
inhibit the generation of two DC precursor cells.
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| Fig 3.
TGF-1 inhibited the expression of Ia and CD86 antigens
and CIITA mRNA in Linc-kit+ HPC cultures
in the presence of GM-CSF + SCF with or without TNF at day 6. (A)
Dose-dependent inhibition on the generation of
Ia+CD86+ cells from murine
Linc-kit+ HPCs stimulated with GM-CSF + SCF + TNF at day 6. The data represent the mean value ± SD of
the percentage and absolute numbers of
Ia+CD86+ cells observed in more than five
experiments. *P < .05 significance as compared with the
cultures without addition of TGF-1. (B)
Ia+CD86+ cells in
Linc-kit+ HPC cultures stimulated with the
indicated various combinations of cytokines. Quads were set up on the
isotype-matched control dot plot. (C) The expression of CIITA mRNA in
Linc-kit+ HPC cultures stimulated with
indicated various combinations of cytokines. The results are
representative of more than three experiments.
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The expression of MHC class II gene is strictly controlled by a
transcription activator CIITA.30,31 In RT-PCR analysis, TGF-1 potently suppressed CIITA mRNA expression in
Linc-kit+ HPCs stimulated with GM-CSF + SCF at day 6 irrespective of TNF addition (Fig 3C), suggesting the
correlation of the inhibitory effect of TGF-1 on DC precursor
differentiation from Linc-kit+ HPC with
suppressed expression of CIITA mRNA.
The inhibitory effect of TGF-1 on mature DC
generation is compromised at day 12 in GM-CSF + SCF + TNF-stimulated
Linc-kit+HPC cultures.
In the presence of GM-CSF + SCF + TNF,
Linc-kit+ HPCs differentiate into mature
DCs at day 12 to 14.9,25 The addition of TGF-1
significantly reduced the appearance of
Ia+CD86+ mature DCs from 36.1% ± 8.7% to 14.4% ± 4.1% in GM-CSF + SCF + TNF-stimulated
cultures at day 12 (Fig 4A and B),
accompanied with reduced capacity to enhance allogenic MLR (P < .05 significance; Fig 4C). In contrast to the inhibitory effect of
TGF-1 on the generation of
CD11b/dullCD11c+ and
CD11b+hiCD11c+ DC precursors at day 6 (Fig 2A),
TGF-1 did not affect the development of
CD11b/dullCD11c+ cells at day 12 (Fig 5) and failed to suppress the
transcription of CIITA gene (Fig 4D) in GM-CSF + SCF + TNF-stimulated Linc-kit+ HPC
cultures. However, the expression of Ia and CD86 antigens was markedly
suppressed on these CD11b/dullCD11c+
cells in the presence of TGF-1 (Fig 5). These results suggest that
the mechanism for TGF-1-mediated inhibitory effect on the generation of nonproliferating
CD11b/dullCD11c+ and
CD11b+hiCD11c+ DC precursors at day 6 may be
distinct from that on the generation of
Ia+CD86+ mature DCs at day 12 in the presence
of TNF. Moreover, these CD11b/dullCD11c+ cells could finally
differentiate into mature DCs in a prolonged cultures (data not shown),
indicating that an alternative DC differentiation pathway, which
differs from CD11b/dullCD11c+ and
CD11b+hiCD11c+ DC precursor cell-mediated ones,
might exist in TGF-1-supplemented cultures.
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| Fig 4.
The effect of TGF-1 on the generation of mature DCs
and the expression of CIITA mRNA in
Linc-kit+ HPC cultures stimulated with
various combinations of cytokines at day 12. (A) The histogram quads,
which were set up on the isotype-matched control dot plot, and (B) the
percentage of Ia+CD86+ cells generated from
GM-CSF + SCF + TNF-stimulated
Linc-kit+ HPCs. The data represent the
mean value ± SD of the percentage of
Ia+CD86+ cells. *P < .05 significance as compared with the cultures without the addition of
TGF-1. The results are representative of more than five experiments.
(C) Allogenic MLR induced by Linc-kit+ HPC
cultures in the presence of the indicated combination of cytokines at
day 12. Results are expressed as the mean ± 1 SD of triplicate
cultures and are representative of three independent experiments. (D)
The expression of CIITA mRNA in various combination of
cytokine-stimulated Linc-kit+ HPCs as
indicated at day 12, which represents three independent experiments.
(*Linc-kit+ HPCs were cultured in
the presence of GM-CSF + SCF + TGF-1 for 12 days and then
recultured in the presence of GM-CSF + TNF for an additional 3 to
5 days.)
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| Fig 5.
TGF-1 inhibited at day 12 the expression of Ia and
CD86 antigens on CD11b/dullCD11c+ cells
derived from cultured Linc-kit+ HPCs
stimulated with the indicated various combinations of cytokines.
Histograms shown in the figures were gated on
CD11b/dullCD11c+ cells. 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.
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TGF-1 inhibits differentiation of
CD11b+hiCD11c+, but not
CD11b/dullCD11c+ DC precursors
into mature DCs.
To examine the effect of TGF-1 on final maturation of DC from
CD11b+hiCD11c+ and
CD11b/dullCD11c+ DC precursors, they
were sorted at day 6, as previously described,9 and then
recultured in the presence of GM-CSF + TNF with or without TGF-1
for an additional 5 days. TGF-1 could significantly inhibit the
generation of Ia+CD86+ mature DCs from
CD11b+hiCD11c+, but not
CD11b/dullCD11c+ DC precursors
(Fig 6) without changing the expression of
CIITA mRNA (data not shown). Consistently, TGF-1 also decreased the capacity of CD11b+hiCD11c+ DC precursor-derived
cells to stimulate allogenic MLR, but did not affect that of the
offspring of CD11b/dullCD11c+ DC
precursor cells (Fig 6).
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| Fig 6.
TGF-1 inhibited DC maturation from
CD11b+hiCD11c+, but not
CD11b/dullCD11c+ DC precursors in the
presence of GM-CSF + TNF. (A) Histograms of quad of
Ia+CD86+ mature DCs were set up on the
isotype-matched control dot plot. (B and C) The capacity of cultured
cells to stimulate allogenic MLR. The experiments are representative of
more than three independent experiments.
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TGF-1 potentiates Linc-kit+
HPCs to differentiate into macrophages with the capacity to
differentiate into LCs.
To better understand the cellular mechanism for the differential effect
of TGF-1 on the generation of nonproliferating DC precursors and
differentiation of mature DCs, Linc-kit+
HPCs were then cultured in the presence of GM-CSF + SCF without the
addition of TNF for 12 days. At day 12, GM-CSF + SCF induced a small
number of Ia+CD86+ mature DCs from
Linc-kit+ HPCs (Figs 4 and 5), whereas
the addition of TGF-1 completely blocked the induction of
Ia+CD86+ mature DCs (Figs 4A and 5) accompanied
with inhibited expression of CIITA mRNA in the same cultures (Fig 4D).
Morphological analyses showed that TGF-1 in combination with GM-CSF + SCF induced differentiation of
Linc-kit+ HPCs solely into
monocyte/macrophage-like cells with large size, many vacuoles in the
cytoplasm, and positive NSE staining, whereas the cell processes and
projections were not observed (Fig 7A, B,
and C). Phenotypically, they expressed high levels of F4/80, CD16/32,
DEC-205, and E-cadherin and low or undetectable levels of Ia, CD86, and
CD40 antigens, complying with monocyte/macrophage phenotype
(Fig 8A). Although these cells expressed
low levels of CD11c and showed the phenotype of
CD11b/dullCD11cdull (Fig 5), they were
active in endocytosis (Fig 9A) and
phagocytosis (Fig 9C), but incapable of enhancing allogenic MLR (Fig
9E). Taken together, all of these results suggest that TGF-1 can
potentiate the differentiation of
Linc-kit+ HPCs into
monocytes/macrophages, but completely blocked the generation of mature
DCs in collaboration with GM-CSF + SCF.
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| Fig 7.
TGF-1 polarized the differentiation of murine
Linc-kit+ HPCs into DCs through the
monocyte/macrophage differentiation pathway. Murine
Linc-kit+ HPCs were first cultured in the
presence of GM-CSF + SCF for 12 days with the addition of TGF-1
(A, B, and C). The cultured cells were then washed twice and recultured
in the presence of GM-CSF + TNF for an additional 3 to 5 days (D,
E, and F). (A and D) Observation by phase-contrast inverted microscope;
(B and E) Giemsa-Wright staining; (C and F) NSE staining. Original
magnifications: for (A) and (D) × 200; for (B), (C), (E), and (F) × 400.
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| Fig 8.
The phenotype of TGF-1-induced
monocytes/macrophages and their offspring of mature DCs. Murine
Linc-kit+ HPCs were first cultured in the
presence of GM-CSF + SCF for 12 days with the addition of TGF-1
(A). These cultured cells were then washed twice and recultured in the
presence of GM-CSF + TNF for an additional 3 to 5 days (B). Cells
were processed for two-color immunofluorescence analyses. Gr-1 and CD40
antigens were examined by FITC-conjugated anti-Gr-1 and CD40 MoAbs.
CD86, F4/80, CD16/32, E-cadherin, and DEC-205 antigens were stained
with uncoupled rat-antimouse MoAbs, followed by staining with
FITC-conjugated antirat IgG. The second color was shown by a
PE-conjugated Ia MoAb. Quads were set up on the isotype-matched control
dot plot. Representative results from three independent experiments are
shown.
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| Fig 9.
The functional maturation of TGF-1-induced
monocytes/macrophages into mature DC-like cells. Murine
Linc-kit+ HPCs were first cultured in the
presence of GM-CSF + SCF for 12 days with the addition of TGF-1 (A
and C). These cultured cells were then washed twice and recultured in
the presence of GM-CSF + TNF for an additional 3 to 5 days (B and
D). A three-color immunofluorescence analysis was performed to show the
capacity of FITC-DX and FITC-Latex uptake by gating on
CD11b/dullCD11c+ cells as
described in Materials and Methods. Solid and dotted lines indicate the
FITC intensity of cells as a control and the test of FITC-DX or
FITC-latex uptake, respectively. (E) TGF-1-induced
monocytes/macrophages ( ) matured into DC-like cells ( ) with the
capacity to potently stimulate allogenic MLR. The results are
representative of three experiments.
|
|
However, when these cells were collected at day 12, washed twice, and
exposed to GM-CSF + TNF for an additional 3 to 5 days, typical
mature DCs developed, as evident with DC aggregates and morphology in
the secondary cultures (Fig 7D and E) and the phenotype expressing high
levels of Ia, CD86, and DEC205 molecules (Fig 8B). Interestingly, all
of these mature DCs expressed high levels of E-cadherin antigen, a
discernible marker for LC,1,10,35,36 indicating that they
are phenotypically LCs (Fig 8B). Functionally, their endocytic (Fig 9B)
and phagocytic activities (Fig 9D) were decreased, whereas the capacity
of stimulating allogenic MLR was significantly enhanced (Fig 9E).
Moreover, most of the mature DCs derived from TGF-1 + GM-CSF + SCF-induced monocytes/macrophages expressed higher levels of E-cadherin
compared with those derived from GM-CSF + SCF-stimulated cells or
GM-CSF + SCF + TNF-induced cells (data not shown), suggesting that
TGF-1 may drive Linc-kit+ HPCs to
differentiate into LC precursors. Interestingly, phenotypic examination
also showed that TGF-1 significantly suppressed the expression of
CCR7 mRNA at day 6 in GM-CSF + SCF-stimulated
Linc-kit+ HPCs irrespective of TNF
addition (Fig 10). Furthermore, TGF-1 inhibited at day 12 the expression of CCR7 mRNA in GM-CSF + SCF-stimulated Linc-kit+ HPCs in the
absence of TNF, indicating that TGF-1 may also play an important
role in the regulation of the localization of LC precursors in situ by
modulating the expression of a chemokine receptor.
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| Fig 10.
The effect of TGF-1 on the expression of E-cadherin
antigen and CCR7 mRNA in cultured
Linc-kit+ HPCs. (A)
Linc-kit+ HPCs were cultured in the
presence of various combinations of cytokines as indicated for 12 days,
followed by washing twice, and were recultured in the presence of
GM-CSF + TNF for an additional 3 to 5 days. These recultured cells
were processed for two-color analyses by staining with a rat-antimouse
E-cadherin MoAb and a PE-labeled mouse antimouse Ia and finally shown
by FITC-conjugated goat-antirat IgG(Fab')2.
Histograms shown in the figures were gated on Ia+hi
cells. Solid and dotted lines indicate the immunofluoresecence
intensity of cells stained with a control MoAb and anti-E-cadherin
antigen, respectively. Representative results from three experiments
are shown. (B) Examination by RT-PCR of CCR7 mRNA in
Linc-kit+ HPCs stimulated with indicated
various combinations of cytokines at day 6 or 12.
|
|
| |
DISCUSSION |
We have recently showed that murine
Linc-kit+ HPCs can differentiate into
mature DCs through bifurcated differentiation pathways of
CD11b/dullCD11c+ and
CD11b+hiCD11c+ DC precursors in response to
GM-CSF + SCF + TNF stimulation.9 In this report, we
first investigated the regulatory effect of TGF-1 on DC generation
in vitro from Linc-kit+ HPCs in the
presence of GM-CSF + SCF + TNF. The results presented here showed
that TGF-1 could potently inhibit the generation of
CD11b/dullCD11c+ DC and
CD11b+hiCD11c+ DC precursors from
Linc-kit+ HPCs stimulated with GM-CSF + SCF + TNF in vitro. These results indicate that TGF-1 is a
negative regulator for the generation of nonproliferating DC precursor
subsets from proliferating DC progenitors, in concordance with its
inhibitory effect on early HPCs.24,37,38
TGF-1 could also suppress DC's maturation from
CD11b+hiCD11c+ DC precursors and
Linc-kit+ HPCs stimulated with GM-CSF + SCF + TNF at late stage (for example, at day 12) based on the
suppressed expression of MHC class II and CD86 molecules and the
reduced capacity of enhancing allogenic MLR. This is substantially
supported by the fact that gene-targeted disruption of TGF-1 results
in markedly enhanced expression of MHC class II antigen and various
autoimmune diseases in mice.23,24 However, the mechanism
for TGF-1-mediated immunosuppression is still an open question.
Several previous reports showed that administration of TGF-1, gene
transfer of TGF-1, or TGF-1-induced "DCs" in vitro could
prolong murine cardiac allograft survival by inhibiting cellular
immunity.39-41 It has recently been demonstrated that many
tumor cells secrete TGF-1 and can activate endogenously produced
latent TGF-1 to bioactive form.42-45 DCs that infiltrate in colon, basal-cell skin cancers,46 and the progressing
melanoma metastases47 lack CD86 and therefore have reduced
T-cell stimulatory activity.46,47 Moreover, the fact that
tumor peptide-pulsed DCs can effectively stimulate the host immune
responses to eradicate the melanoma cells48 alternatively
suggests that endogenously functional disorder of DCs may partially
contribute to the tumorigenicity. Several soluble factors have been
implicated in defective DC maturation in cancer, including vascular
endothelial growth factor (VEGF)1,49 and
IL-10.1,48 Our findings further suggest that TGF-1 may also play an important role in negatively regulating immune responses in vivo by modulating DC's development and functions.
Phenotypic examination also demonstrated that murine
Linc-kit+ HPC-derived DCs and DC
precursors mainly expressed CCR1 and CCR7 (data not shown).
Unexpectedly, TGF-1 suppressed the expression of CCR7 mRNA in
Linc-kit+ HPCs stimulated with GM-CSF + SCF + TNF at day 6 and in GM-CSF + SCF-stimulated
Linc-kit+ HPCs either at day 6 or 12, including LC precursors. Because migration of DCs is thought to be
regulated by the interaction of chemokine and chemokine
receptor.50,51 Disconnecting such migration of DCs may
prevent antigen-specific immune responses.1 It may prove to
be valuable to elucidate whether TGF-1 might regulate the migration
of DC and DC precursors in vivo through modulating the expression of
chemokine receptors.
Our results appear in contrast to previous investigations that TGF-1
enhances the generation of mature DCs from human CD34+ HPCs
in serum-free cultures stimulated with GM-CSF + TNF.19 A distinct culture system and HPC species may
simply account for the discrepancy. However, DCs are heterogeneous
populations originating from distinct differentiation
pathways,1-9,52-55 and the maturation of DCs can be
divided into at least three stages based on the expression of MHC class
II and other costimulatory molecules.56,57 Moreover, many
other reports demonstrate that TGF-1 shows different effects on
proliferation and differentiation of HPCs, depending on the
differentiation state of HPCs and supplemented
cytokines.24,37,38,58 We presume that the apparent
discrepancy might be related to the distinct effect of TGF-1 on the
generation of DC precursors from HPCs and differentiation of DC
precursors into mature DCs under different culture conditions.
Accumulating evidence suggests that monocytes/macrophages can
differentiate into LCs.1,10,59 Decreased numbers of LCs and
monocytes/macrophages were previously documented in the op/op mouse, a null mutant of the M-CSF gene.59 When combined
with GM-CSF + IL-4, TGF-1 can drive human peripheral monocytes to differentiate into LCs,10 further supporting the notion of
the tight connection of monocytes/macrophages with the ontogeny of LCs.
We observed that TGF-1 could not directly induce the differentiation and maturation of LC from Linc-kit+ HPCs
stimulated with GM-CSF + SCF in the absence of TNF, but indeed
induced the generation of monocytes/macrophages at day 12 to 14, as
evident in morphology and phenotype such as the expression of high
levels of F4/80, but low or undetectable levels of Ia, CD86, and CD40
molecules. Interestingly, these cells expressed high levels of DEC-205
and E-cadherin antigens (Fig 8A). They were further able to
differentiate into mature LCs expressing high levels of E-cadherin and
other mature DC markers in response to GM-CSF and TNF, consistent
with previous described role of endogenous TGF-1 in LC
development.20 It is likely that the TGF-1-induced
monocytes/macrophages represent LC precursors. Therefore, we
hypothesize that TGF-1 may potentiate HPCs to differentiate into
monocytes/macrophages that will be anchored by increasing the
expression of E-cadherin protein in situ in the epidermis, where they
differentiate into mature LCs in response to stimuli, such as GM-CSF,
TNF, and IL-4, in inflammatory reactions. Accordingly, locally
produced TGF-1 itself may maintain the DC precursors at immature
stage by inhibiting the expression of MHC class II and costimulatory
molecules, which plays critical actions in regulating antigen
processing and maintaining immune responses.
It is believed that various DC subsets may play distinct roles in
immune responses.1,2,5-8,60 Human mature DCs derived from
CD1a+CD14 and
CD1aCD14+ DC precursors display distinct
role in stimulating humoral immune responses and regulating the
secretion of Igs, respectively.5,60 Based on the phenotype
and differentiation capacity, murine
CD11b+hiCD11c+ DC precursors may correspond to
human CD1aCD14+ DC precursors, whereas
CD11b/dullCD11c+ DC precursor may
correspond to human CD1a+CD14 DC
precursors.4,5,9 TGF-1 inhibited the generation of CD11b/dullCD11c+ DC and
CD11b+hiCD11c+ DC precursors from
Linc-kit+ HPCs and mature DCs from
CD11b+hiCD11c+ DC precursors. In contrast,
TGF-1 polarizes the development of LC-like DCs expressing high
levels of E-cadherin, c-fms (data not shown), and NSE activity from
Linc-kit+ HPCs through the
monocyte/macrophage differentiation pathway, which obviously differ
from the phenotype of CD11b/dullCD11c+
DC and CD11b+hiCD11c+ DC precursor-derived
mature DCs9 (Fig
11). Such polarization effect of TGF-1 on DC generation
may play important pathophysiological roles in regulating various
immune responses.
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| Fig 11.
A schematic DC differentiation model in vitro from
Linc-kit+ HPCs and the regulating role of
TGF-1. HPCs develop into mature DCs through four stages:
proliferating DC progenitor cells, nonproliferating DC precursors,
antigen capturing immature DCs, and mature DCs. The cytokine
combination of GM-CSF + SCF + TNF can induce the generation of
mature DCs from murine Linc-kit+ HPCs
through two unrelated differentiation pathways:
CD11b/dullCD11c+ and
CD11b+hiCD11c+ DC precursors that can be
clearly identified at day 6 of culture. In response to GM-CSF + TNF, both the DC precursor subsets can independently differentiate
at day 10 to 14 into mature DCs with distinct phenotype based on the
expression of c-fms mRNA, NSE activity, and E-cadherin. TGF-1
significantly inhibited the generation of
CD11b/dullCD11c+ and
CD11b+hiCD11c+ DC precursors from GM-CSF + SCF + TNF-stimulated Linc-kit+
HPCs at day 6 of culture. TGF-1 could also suppress DC maturation
from CD11b+hiCD11c+, but not
CD11b/dullCD11c+ DC precursors at day 12 to 14. In collaboration with GM-CSF + SCF, TGF-1 induced
Linc-kit+ HPCs to differentiate solely
into monocytes/macrophages. These cells could further differentiate at
day 15 to 17 of culture into LC-like DCs expressing high levels of
E-caderin, abundant c-fms, and NSE activity, which obviously differs
from CD11b/dullCD11c+ and
CD11b+hiCD11c+ DC precursor-derived mature
DC subsets.
|
|
The molecular mechanisms for the polarization effect of TGF-1 on DC
differentiation remain elusive. TGF-1 can inhibit the expression of
CIITA in several types of cells61,62 by suppression of the
basal promoter.62 TGF-1 significantly inhibited the expression of CIITA mRNA in GM-CSF + SCF-treated
Linc-kit+ HPCs at day 6, irrespective of
addition of TNF or at day 12 in the absence of TNF, in parallel
to its suppressing effect on the generation of nonproliferating
CD11b/dullCD11c+ and
CD11b+hiCD11c+ DC precursors in the same
cultures. Moreover, gene-targeted disruption of CIITA in mice impairs
the expression of MHC class II molecules on DCs and results in
functional incapacity of DCs to stimulate allogenic MLR.31
These data suggest that the expression of CIITA is tightly related to
the differentiation of DC precursors and functionally mature DCs from
HPCs. Presumably, the suppressed expression of CIITA may be partially
responsible for TGF-1-induced inhibitory effect on the generation
of CD11b/dullCD11c+ and
CD11b+hiCD11c+ DC precursors. However, TGF-1
failed to inhibit the transcription of CIITA gene at day 12 in GM-CSF + SCF-treated Linc-kit+ HPCs in the
presence of TNF and in CD11b+hiCD11c+ DC
precursors. But it suppressed DC maturation in these cultures by
decreasing the expression of Ia and CD86 antigens. Because the
expression of MHC class II molecule is not only strictly regulated by
CIITA at the transcriptional level, but also regulated at the posttranscriptional level, including translation, synthesis,
translocation, and recycling of MHC class II
molecules,30,31,56,57 it is conceivable that TGF-1 may
also confer its inhibitory effect on DC maturation at the
posttranscriptional level. This may help us to gain
insight into the molecular mechanisms for development of DC from early HPCs.
| |
ACKNOWLEDGMENT |
The authors express our sincere gratitude to Dr R.M. Steinman for his
kind gift of MoAbs to DEC-205 (NLDC145) and 33D1 and to Dr T. Sudo for
his generous gift of an anti-c-kit MoAb, GM-CSF, and SCF. We also
greatly appreciate Dr C. Vestergaard for helpful discussion.
| |
FOOTNOTES |
Submitted May 21, 1998; accepted October 15, 1998.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
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.
| |
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Abstract
Introduction