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Departments of
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Cell Processing and
Clinical Immunology and AIDS Research Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan; and
Japanese Red Cross Central Blood Center, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(MIP-3) (for CCR-6), or stromal cell-derived growth
factor-1 (for CXCR-4). Ligation by TNF- resulted in
down-modulation of cell surface expressions of CCR-1, CCR-3, CCR-5,
CCR-6, and CXCR-4, and the chemotaxis for RANTES, MIP-3, and stromal
cell-derived growth factor-1, whereas this stimulation up-regulated
the expression of CCR-7 and the chemotactic ability for MIP-3ß.
Stimulation of mature DCs with TGF-ß1 also enhanced TNF--induced
down-regulation of the expressions of CCR-1, CCR-3, CCR-5, CCR-6, and
CXCR-4, and chemotaxis to their respective ligands, while this
stimulation suppressed TNF--induced expression of CCR-7 and
chemotactic migratory ability to MIP-3ß. Our findings suggest that
TGF-ß1 reversibly regulates chemotaxis of DCs via regulation of
chemokine receptor expression. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Characterization of DCs is difficult because they represent only a small subpopulation that includes interdigitating reticulum cells in lymphoid organs, blood DCs, Langerhans cells in the epidermis of the skin, and dermal DCs (1). Previously, an in vitro culture system revealed that DCs originate from CD34+ pluripotent hemopoietic progenitor cells (HPCs) in the bone marrow (BM) and cord blood via myeloid lineage cells in human and murine models (7, 8, 9, 10, 11, 12, 13, 14, 15, 16), and some DCs develop from thymic precursors via lymphoid lineage cells in murine system (17).
Chemokines are extensively involved in inflammatory/immunological
responses due to their unique ability to recruit selective leukocyte
subsets (18). Chemokines have been implicated in
regulation of normal leukocyte recirculation and homing, and also in
certain physiological and pathogenic processes, including hemopoiesis,
angiogenesis, allergy, autoimmune diseases, and viral infectious
diseases (18). Chemokines are a group of 
70–90 amino
acids, structurally related polypeptides, most of which contain four
conserved cysteine residues in their primary amino acid sequence
(18). There are two major groups: the CXC chemokines in
which the two NH2-terminal cysteines are
separated by a single amino acid, and the CC chemokines, in which
the two NH2-terminal cysteines are adjacent. A
third type of chemokine, represented by lymphotactin, contains only two
of the four conserved cysteines (18).
The specific effects of chemokines on the target cell types are
mediated by a family of single-chain, seven-helix membrane-spanning
receptors coupled to heterotrimeric guanine nucleotide-binding protein
(G protein) (GPCR), which consists of a Gi, Gß, and G
subunit
complex (18). Ligand specificities of 14 chemokine
receptors have been identified; five of the receptors are specific for
CXC chemokines (CXCR1–5) (18), eight of them are specific
for CC chemokines (CCR1–8) (18, 19, 20), and the Duffy Ag
receptor binds both CXC and CC chemokines (21). In
addition, distinct chemokines appear to act on more than one receptor
type in vitro (18).
There is increasing interest in the potential role of chemokines and their respective receptors in the biological properties of DCs to clarify the mechanism underlying DC-mediated regulation of immune/inflammatory responses. Previous studies have shown that several chemokine receptors are expressed on some DCs and their progenitor cells at the transcriptional level (22, 23, 24, 25, 26, 27). Recent studies have shown that the chemotactic migratory properties in response to certain chemokines are strictly regulated in the development of DCs from their progenitor cells, and these regulatory mechanisms have been potentially implicated in mediating the trafficking of DCs and their progenitor cells from blood to tissues and then to lymph nodes, where they form a close association with TCs in the process of Ag presentation (26, 27).
TGF-ß1, which is a cytokine produced by various types of cells, is a pleiotropic cytokine that has growth-modulatory, immunosuppressive, and inflammatory activities (28, 29, 30). Previous studies have shown that TGF-ß1 is involved in the generation of DCs and Langerhans cells from their progenitor cells (31, 32, 33, 34). Furthermore, Ogata et al. (35) have recently reported that TGF-ß1 not only inhibited the expression of CCR-7 in DCs and DC precursors derived from HPCs, but it also inhibited the migration of these cells in response to macrophage-inflammatory protein (MIP)-3ß in murine models. However, the role of TGF-ß1 in the regulation of chemotaxis of human DCs remains unclear.
We have recently reported that CCR-1, CCR-3, CCR-5, and CXCR-4 are
constitutively expressed on the cell surface of human peripheral blood
monocyte-derived immature DCs (iDCs) (36). Furthermore,
mAb to either CCR-1 or CCR-3, but not CCR-5 and CXCR-4, abolished the
chemotactic migratory ability of monocyte-derived iDCs as well as the
ability of these cells to activate allogeneic TCs to proliferate
and secrete IFN-, indicating that CCR-1 and CCR-3 specifically
regulate interactions between TCs and iDCs via chemotactic migratory
events during Ag presentation (36).
In this study, we examined the potential roles of TGF-ß1 in the regulation of chemotactic migratory properties of human monocyte-derived DCs via chemokine receptors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The medium used throughout was RPMI 1640 supplemented with 2 mM
L-glutamine, 50 µg/ml streptomycin, 50 U/ml penicillin,
and 10% heat-inactivated FCS. GM-CSF was kindly provided by Kirin
Brewery (Tokyo, Japan). IL-4, IL-10, TNF-, TGF-ß1, RANTES,
MIP-1, eotaxin, stromal cell-derived factor (SDF)-1, MIP-3,
and MIP-3ß were purchased from PeproTech (London, U.K.). FITC-labeled
dextran (FITC-DX) and lucifer yellow (LY) were purchased from Molecular
Probes (Eugene, OR). A mAb to IL-10 was purchased from PharMingen (San
Diego, CA). The preparations of mAbs to CCR-1 and CCR-3 (IgG1
isotype), and the specific recognition of these mAbs for the respective
CCRs were described previously (36).
In vitro generation and culture of human DCs
DCs were generated from PBMCs, as described previously (9, 10), with some modifications (36, 37, 38). Briefly,
PBMCs were obtained from 30 ml of leukocyte-enriched buffy coat from
healthy donors by centrifugation with Ficoll-Hypaque (Pharmacia Fine
Chemicals, Uppsala, Sweden), and the light density fraction from the
42.5–50% interface was recovered. The cells were resuspended in
culture medium and allowed to adhere to six-well plates (Costar,
Cambridge, MA). After 2 h at 37°C, nonadherent cells were
removed and adherent cells were collected, then subsequently negatively
selected with anti-CD2 mAb-conjugated immunomagnetic beads (Dynal,
Oslo, Norway) and anti-CD19 mAb-conjugated immunomagnetic beads
(Dynal) to deplete CD2+ cells and
CD19+ cells, according to the manufacturer’s
instructions. The resultant cells (>95% CD14+
cells) were used as monocytes and cultured in 3 ml of medium
supplemented with GM-CSF (50 ng/ml) and IL-4 (250 ng/ml). After 7 days
of culture, iDCs were harvested (36, 37, 38). For preparation
of mature DCs (mDCs) from iDCs, the cells were subsequently cultured
with TNF- (50 ng/ml) for another 4 days (37, 38). These
cell populations exhibited the typical phenotype of DCs, as described
previously (36, 37, 38). Cell differentiation was monitored by
light microscopy, and the resultant cells were used for subsequent
experiments.
Preparation and culture of TCs
TCs were isolated from monocyte-depleted cell population by E-rosetting (39), and TC preparations were typically >95% pure, as indicated by anti-CD3 mAb (Becton Dickinson, Mountain View, CA) staining.
Stimulation of DCs
iDCs or mDCs (107) were unstimulated or stimulated with various concentrations of TGF-ß1 or IL-10 (1–100 ng/ml) in the presence or absence of 1 µg/ml control mouse IgG (Sigma, St. Louis, MO) or anti-IL-10 mAb for the indicated number of days (1–5 days). The cells were washed twice with cold PBS, and used for subsequent experiments.
Flow cytometry
For surface marker analysis, iDCs or mDCs were cultured with the following mAbs conjugated to FITC or PE for direct fluorescein: CD83 (Coulter Immunology, Hialeah, FL); HLA-DR (Becton Dickinson, Mountain View, CA); CD40, CD86, and CCR-5 (all from PharMingen); CCR-6 (R&D Systems, Minneapolis, MN). The cells were also stained with the corresponding FITC- or PE-conjugated isotype-matched control mAb (all from Becton Dickinson). For indirect staining, the cells were incubated with biotin-conjugated anti-CCR-1 mAb, biotin-conjugated anti-CCR-3 mAb, or biotin-conjugated anti-CXCR-4 mAb (PharMingen) for 30 min at 4°C, washed twice with cold PBS, and subsequently stained with FITC-conjugated avidin (Becton Dickinson) for 30 min at 4°C. Thereafter, the cells were washed twice, and suspended in PBS containing 0.2 µg/ml propidium iodide (Sigma) to exclude dead cells. Analysis of fluorescence staining was performed with a FACScalibur flow cytometer (Becton Dickinson) and CELLQuest Software. The cell surface expression levels in the flow cytometry profiles are expressed as the mean fluorescence intensity (MFI) indices.
Assay for chemotaxis
The in vitro migration of iDCs or mDCs was assessed in a 24-well
Transwell cell culture chamber (Costar), as described previously
(36, 38). In brief, 8-µm-pore-size polycarbonate filters
were precoated with 5 µg of gelatin (WAKO, Osaka, Japan) in a volume
of 50 µl on the lower surface, and dried overnight at room
temperature. The coated filters were washed in PBS and then dried
immediately before use. DCs were pretreated with or without 1 µg/ml
of the mAbs to CCR-1, CCR-3, CCR-5, CXCR-4, or control mouse IgG
(Sigma) for 30 min at 37°C, and 100 µl of the cell suspension
(106) was added to the upper compartment of the
chamber. RANTES, MIP-1, eotaxin, SDF-1, MIP-3, or MIP-3ß
(1–100 ng/ml) diluted in serum-free culture medium was loaded in the
upper compartment and/or the lower compartment. After a 2-h incubation,
the filters were fixed with methanol and stained with hematoxylin and
eosin (all from WAKO, Osaka, Japan). The cells on the upper surface of
the filters were removed by wiping with cotton swabs. The cells that
had migrated to various areas of the lower surface were manually
counted under a microscope at a magnification of x200, and each assay
was performed in triplicate. The data are expressed as number of
migrated cells/high power fields.
Endocytosis assay with FITC-DX and LY
The methods used to determine the endocytotic activity of in vitro generated DCs have previously been described (37, 38). Briefly, FITC-DX or LY was added to a final concentration of 1 mg/ml to the cells, and the cells were cultured for 60 min at 37°C. After incubation, cells were washed four times with ice-cold PBS and analyzed by flow cytometry, as described above.
Mixed leukocyte reaction
A total of 105 responding T cells from an unrelated individual (allogeneic MLR) were cultured in 96-well flat-bottom microplates (Costar) with different numbers (102–5 x 104) of stimulator cells. Thymidine incorporation was measured on day 5 by an 18-h pulse with [3H]thymidine (1 µCi/well; sp. act., 5 Ci/mmol; Amersham Life Science, Buckinghamshire, U.K.).
Reverse-transcriptase PCR
RNA from each sample (5 x 106) was isolated using Trizol LS reagent (Life Technologies, Gaithersburg, MD). The first strand cDNA kit (SuperScript Preamplification System; Life Technologies) was used to make cDNA (20 µl) from 5 µg of each RNA. Amplification of each cDNA (1 µl) was performed with a SuperTaq Premix kit (Sawady Technology, Tokyo, Japan) using specific primers, as follows: CCR-1, 5'-TCC TCA CGA AAG CCT ACG AGA GTG GAA GC-3' and 5'-CCA CGG AGA GGA AGG GGA GCC ATT TAA C-3'; CCR-3 (23), 5'-TTT GGT GTC ATC ACC AGC AT-3' and 5'-TCA TGC AGC AGT GGG AGT AGG-3'; CCR-5 (23), 5'-GGT GGA ACA AGA TGG ATT AT-3' and 5'-CAT GTG CAC AAC TCT GAC TG-3'; CCR-6 (27), 5'-ATT TCA GCG ATG TTT TCG ACT C-3' and 5'-GGA GAA GCC TGA GGA CTT GTA-3'; CCR-7 (27), 5'-GAT TAC ATC GGA GAC AAC ACC-3' and 5'-TAG TCC AGG CAG AAG AGT CG-3'; CXCR-4, 5'-CTG AGA AGC ATG ACG GAC AAG TAC AGG CT-3' and 5'-CAG ATG AAT GTC CAC CTC GCT TTC CTT TGG-3'. Specific primers for ß-actin (CLP, San Diego, CA) were also used for amplification. The reaction mixture was subjected to 35 cycles of PCR with the following conditions: CCR-1 and CXCR-4, 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min. CCR-3 and CCR-5, 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min. CCR-6 and CCR-7, 94°C for 1 min, 61.5°C for 2 min, and 72°C for 3 min. PCR products were analyzed by electrophoresis through 2% agarose gels and visualized under UV light after ethidium bromide staining.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The chemotactic migratory property of DCs is thought to be crucial
for the regulation of immune responses, as well as inflammation and
autoimmune diseases. However, little is known about the regulation of
chemotaxis of DCs. Previous studies have shown that TGF-ß1 is
involved in the development of DCs as well as Langerhans cells from
their progenitor cells (31, 32, 33, 34). We have recently reported
that human monocyte-derived iDCs constitutively express CCR-1, CCR-3,
CCR-5, and CXCR-4 on their cell surface (36). To examine
the effect of TGF-ß1 on the expression of these CC and CXCR in iDCs,
the cells were unstimulated or stimulated with TGF-ß1 (10 ng/ml) for
3 days, and the expressions of these chemokine receptors were analyzed
by flow cytometry with respective mAbs. Stimulation of iDCs with
TGF-ß1 resulted in the enhancement of the expression levels of CCR-1,
CCR-3, CCR-5, and CXCR-4 on their cell surfaces as compared with those
of unstimulated iDCs (Fig. 1
A).
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, treatment of iDCs with
TGF-ß1 induced the expressions of these chemokine receptors in a
dose-dependent manner.
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shows that the
expressions of these chemokine receptors were increased during the
first 3 days, whereas they decreased on day 5.
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To examine the effect of TGF-ß1 on the chemotaxis of iDCs in
response to CC and CXC chemokines, the migratory responses of iDCs,
which were unstimulated or stimulated TGF-ß1, to RANTES, MIP-1,
eotaxin, or SDF-1 using a Transwell cell culture chamber were
studied. As shown in Fig. 4A,
iDCs migrated in response to these chemokines, and TGF-ß enhanced
these chemotactic migrations. The migratory response to these
chemokines by these iDCs was chemotactic and not due to chemokinesis
because migration was not observed in the absence of a chemokine
gradient (Fig. 4A).
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. As shown in Fig. 4
B, iDCs migrated in response to
RANTES or SDF-1 in a dose-dependent manner, and TGF-ß1 enhanced
these chemotactic migrations. In addition, RANTES- or SDF-1ß-induced
chemotaxis of iDCs was enhanced by TGF-ß1 in a dose-dependent fashion
(Fig. 4C).
To address the role of chemokine receptors in the TGF-ß1-induced
enhancement of the chemotaxis of iDCs, effect of mAbs to chemokine
receptors on the chemotactic migration of unstimulated and
TGF-ß1-stimulated iDCs to RANTES or SDF-1 was examined.
Table I shows that both the
anti-CCR-1 mAb and anti-CCR-3 mAb inhibited chemotactic
migration of unstimulated and TGF-ß1-stimulated iDCs to RANTES,
whereas anti-CCR-5 exhibited a slight suppression. The combinations
of mAbs to CCR-1, CCR-3, and CCR-5 exhibited greater inhibition on the
migratory capacity of these cells to RANTES than those of each mAb to
CCRs. On the other hand, anti-CXCR-4, but not mAbs to CCRs,
suppressed SDF-1-induced chemotactic migratory responses of these
cells, whereas this mAb did not suppress RANTES-induced chemotaxis of
these cells.
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Sozzani et al. (40) have previously reported that
IL-10 enhanced the expression of CCR-1, CCR-2, and CCR-5 in human
monocytes at the transcriptional level, and these phenonena were
involved in the enhanced chemotactic migratory capacity for monocyte
chemotactic protein-1 and MIP-1ß. We have also reported that
stimulation of iDCs with IL-10 resulted in up-regulation of their
chemotactic migratory ability to RANTES (38). On the other
hand, TGF-ß1 reportedly enhanced the ability of macrophages to
produce IL-10 in murine systems (28, 29). To examine
whether IL-10 mediates TGF-ß1-induced expression of CC and CXCR, iDCs
were unstimulated or stimulated with IL-10 or TGF-ß1 in the presence
or absence of anti-IL-10 mAb (Table II). Stimulation of iDCs with IL-10
resulted in increased expressions of CCR-1, CCR-3, CCR-5, and CXCR-4,
whereas anti-IL-10 mAb suppressed them. On the other hand,
anti-IL-10 mAb did not affect TGF-ß1-induced expression of CC
and CXCR.
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-induced down-modulation of the
expressions of CC and CXCR and the chemotaxis to their ligands
This study shows that TGF-ß1 exhibited the ability to
up-regulate the cell surface expressions of CCR-1, CCR-3, CCR-5, and
CXCR-4 in iDCs. To examine whether TGF-ß1 affects the transcription
of these chemokine receptors, iDCs were unstimulated or stimulated with
TGF-ß1, and the expressions of their mRNA were analyzed by RT-PCR. As
shown in Fig. 5B, the
transcripts of these chemokine receptors were enhanced by TGF-ß1 as
compared with those of unstimulated iDCs, and these results were
paralleled by their cell surface expressions (Fig. 5A).
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induces the development of iDCs into mDCs, which is associated
with down-modulation of expressions of several chemokine receptors at
the transcriptional levels and chemotactic migratory ability (24, 26, 27). As shown in Fig. 5, TNF- induced down-regulation of
transcripts and products on the cell surfaces of these chemokine
receptors, and these results were consistent with previous reports
(24, 26, 27). We also examined whether TGF-ß1
up-regulates the expressions of CCR-1, CCR-3, CCR-5, and CXCR-4 in mDCs
(Fig. 5). Treatment of mDCs with TGF-ß1 resulted in an enhancement of
the transcripts and products on the cell surfaces of these chemokine
receptors as compared with those of mDCs, and the expression levels
were similar to those of unstimulated iDCs.
We further examined the effect of TGF-ß1 on chemotaxis of mDCs to
RANTES and SDF-1 (Fig. 6). The
chemotactic migratory abilities of mDCs to RANTES and SDF-1 were
lower than those of iDCs. On the other hand, TGF-ß1 increased the
ability of mDCs to migrate for RANTES and SDF-1, and these abilities
were comparable with those of unstimulated iDCs.
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We examined the influence of TGF-ß1 on phenotypic changes in
iDCs and mDCs. As shown in Fig. 7A, TGF-ß1 had little or no
effect on the cell surface expressions of CD40, CD86, and HLA-DR in
iDCs. Furthermore, TNF--induced activation/maturation-associated
enhancement of the cell surface expressions of CD40, CD83, CD86, and
HLA-DR was not affected by TGF-ß1 in mDCs.
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B). The endocytic ability of
TGF-ß1-stimulated iDCs was similar to that of unstimulated iDCs.
Moreover, TNF--induced mDCs exhibited lower endocytic ability than
iDCs, as reported previously (37, 38), while TGF-ß1 had
no effect in mDCs.
To examine whether TGF-ß1 affects the allogeneic TC-stimulatory
abilities of iDCs and mDCs, allogeneic TCs were cultured with various
numbers of these cells, and the proliferative responses were measured
on day 5. Fig. 7C shows that iDCs stimulated allogeneic TCs
to proliferate in a dose-dependent fashion, and the allostimulatory
capacity of mDCs was more potent than that of iDCs. On the other hand,
TGF-ß1 had little or no effect on the abilities of iDC and mDCs to
stimulate allogeneic TC proliferation as compared with unstimulated
cells.
TGF-ß1 induces the expression of CCR-6 and chemotaxis for
MIP-3 in iDCs and mDCs
Chan et al. (41) have recently reported that
the transcript of CCR-6 was expressed in iDCs, whereas its product on
the cell surface was not observed in these cells. On the other hand,
Yang et al. (42) have recently shown that iDCs generated
from monocytes cultured with GM-CSF, IL-4, and TGF-ß1 expressed both
CCR-6 mRNA and protein on the cell surface, and these cells responded
to MIP-3. We therefore tested the effect of TGF-ß1 on expression
of CCR-6 in iDCs and mDCs (Fig. 8,
A and B). The transcriptional expression of CCR-6
was observed in iDCs, whereas the cell surface expression of CCR-6 was
not detected. Stimulation of iDCs with TGF-ß1 resulted in the
increased expressions of the transcript and the product of CCR-6. On
the other hand, the transcriptional and cell surface expression od
CCR-6 were decreased in mDCs, while TGF-ß1 stimulated mDCs to express
CCR-6.
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. Fig. 8C shows that
iDCs did not respond to MIP-3, whereas stimulation of iDCs with
TGF-ß1 enhanced this migration. On the other hand, mDCs failed to
migrate in response to MIP-3, while treatment of mDCs with TGF-ß1
induced this migration.
TGF-ß1 suppresses TNF--induced expression of CCR-7 and
chemotaxis for MIP-3ß in mDCs
Previous studies have shown that CCR-7 is exclusively
expressed in mDCs at the transcriptional level, and these cells migrate
to MIP-3ß via CCR-7 (26, 27). We therefore tested the
effect of TGF-ß1 on expression of the transcript of CCR-7 in mDCs
(Fig. 9A). The transcriptional
expression of CCR-7 was observed in mDCs, but not in iDCs. On the other
hand, TGF-ß1 suppressed this expression in mDCs. We also
examined the effect of TGF-ß1 on the chemotactic migratory ability of
mDCs in response to MIP-3ß. Fig. 9B shows that mDCs
migrated in response to MIP-3ß, while treatment of mDCs with TGF-ß1
suppressed this migration.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The transcriptional and cell surface expressions of CCR-1, CCR-5, and
CXCR-4 were previously shown in various types of human DCs
(22, 23, 24, 26, 36, 41). However, there are conflicting
reports about the expression of CCR-3 in these cells (22, 23, 24, 41). In accordance with previous reports (23, 24, 41), we showed that iDCs constitutively expressed the
transcripts and products on the cell surface of CCR-3 as well as CCR-1,
CCR-5, and CXCR-4 (
Figs. 1–3, 5, and 6). Furthermore, we observed that
iDCs migrated to their respective ligands such as RANTES (for CCR-1,
CCR-3, and CCR-5), MIP-1 (for CCR-1, CCR-2, and CCR-5), eotaxin (for
CCR-3), and SDF-1 (for CXCR-4) (Fig. 4). These results suggest that
these CC and CXCR may be functionally expressed on the cell surfaces
of iDCs.
We showed that stimulation of iDCs with TGF-ß1 enhanced the cell
surface expressions of CCR-1, CCR-3, CCR-5, and CXCR-4 (
Figs. 1–3),
and this stimulation up-regulated the chemotactic migratory ability of
iDCs for RANTES, MIP-1, eotaxin, and SDF-1 (Fig. 4). Furthermore,
mAbs to CCR-1, CCR-3, and CCR-5 significantly inhibited the chemotaxis
of TGF-ß1-stimulated iDCs as well as unstimulated iDCs in response to
RANTES (Table I). We also demonstrated that anti-CXCR-4 mAb
suppressed the SDF-1-induced chemotaxis of these cells (Table I).
These results suggest that the TGF-ß1-induced up-regulation of
chemotaxis of iDCs to CC and CXC chemokines may be mediated by
increased expression of their respective receptors.
It has been shown that TGF-ß1 stimulates macrophages to produce
IL-10, and several biological effects of TGF-ß1 are mediated by IL-10
in murine systems (28, 29). We showed that mAb to IL-10
failed to abrogate TGF-ß1-induced enhancement of the cell surface
expressions of CCR-1, CCR-3, CCR-5, and CXCR-4 in iDCs (Table I). These
results suggest that TGF-ß1-induced enhancement of the expressions of
CCR-1, CCR-3, CCR-5, and CXCR-4 is not mediated by IL-10. Thus,
TGF-ß1 may directly regulate the expressions of these chemokine
receptors in iDCs.
Greaves et al. (25) have previously reported that the
transcript of CCR-6 was expressed in CD34+
HPC-derived DCs, whereas this expression was undetectable in
monocyte-derived DCs. In contrast, recent studies have shown that the
transcriptional expression of CCR-6 was observed in monocyte-derived
iDCs (41, 42). In accordance with previous reports
(41, 42), the transcript of CCR-6 was constitutively
expressed in monocyte-derived iDCs (Fig. 8A), although its
cell surface expression was not detected in these cells (Fig. 8B). Furthermore, stimulation of iDCs with TGF-ß1 induced
the enhancement of the expression of CCR-6 transcript and product (Fig. 8, A and B), and these cells responded to
MIP-3 (Fig. 8C). These results suggest that
TGF-ß1-induced posttranscriptional regulation of CCR-6 may lead to
its functional cell surface expression iDCs.
The expressions of CCR-1, CCR-3, CCR-5, and CCR-6 were detectable on
the cell surface (Figs. 5A and 8B), whereas
little or no transcriptional expression of these CCRs was observed in
mDCs (Figs. 5B and 8A). The reason that the
levels of transcriptional expression of these CCRs were not completely
consisted with the levels of their cell surface expressions remains
unclear, but one possible explanation is that the kinetics of decreased
cell surface expressions of these CCRs may not be different from those
of their transcriptional expressions. Alternatively, other molecular
mechanism(s) as well as the transcriptional regulation may be involved
in the reduced cell surface expressions of these CCRs
(43). Further study will be needed to examine this
possibility.
We showed that stimulation of mDCs with TGF-ß1 resulted in increased
expression of the transcripts and products on the cell surfaces of
CCR-1, CCR-3, CCR-5, CCR-6, and CXCR-4 compared with unstimulated mDCs
(Fig. 6). Furthermore, the chemotactic migratory abilities of
TGF-ß1-stimulated mDCs to RANTES, MIP-3, and SDF-1 were more
potent than those of mDCs (Fig. 7). These findings suggest that
TGF-ß1 may also enhance the chemotaxis to RANTES, MIP-3, and
SDF-1 via up-regulation of the expressions of CCR-1, CCR-3, CCR-5,
CCR-6, and CXCR-4 in mDC. On the other hand, the transcriptional
expression of CCR-7 was suppressed by TGF-ß1 in mDCs, and this
expression was associated with their ability to migrate to MIP-3ß
(Fig. 9). These findings suggest that TGF-ß1 may impair the
chemotactic migratory ability of mDCs for MIP-3ß via down-regulated
expression of CCR-7. In contrast to TGF-ß1-mediated regulation of CC
and CXCR in human monocyte-derived DCs in our experiments, Ogata et al.
(35) have recently reported that the expression of CCR-7
transcript was inhibited in TGF-ß1-treated murine BM-derived DCs when
GM-CSF plus TNF--cultured HPCs were treated with TGF-ß1 for the
first 6–7 days, but not at days 13–14, whereas the transcriptional
expression of CCR-1 was not affected by TGF-ß1 in these cells. This
discrepancy might be due to species differences.
It has been shown that TGF-ß1 negatively modulates several functions
of various cell types (28, 29). Lee et al.
(30) have previously reported that TGF-ß1-transduced
murine BM-derived DCs exhibited lower allogeneic TC stimulatory ability
than control vector-transduced cells, although these types of cells
expressed similar expression levels of MHC products and
adhesion/costimulatory molecules. We showed that TGF-ß1 did not
affect phenotypes and functions of iDCs and mDCs with respect to the
abilities for the endocytosis and the allogeneic TC stimulation (Fig. 8). Furthermore, we observed that TC proliferation was suppressed when
allogeneic TCs were cultured with iDCs or mDCs in the presence of
TGF-ß1 (data not shown), suggesting that TGF-ß1 may not directly
regulate Ag-processing/presenting functions of monocyte-derived DCs,
although TGF-ß1 may suppress DC-mediated activation of TCs. These
phenomena led us to hypothesize that TGF-ß1 may specifically regulate
the chemotactic migratory responses of iDCs and mDCs by regulating the
expression of chemokine receptors.
The role of TGF-ß1 in the trafficking of human DCs in vivo remains unclear.
Recently, the responsiveness of iDCs to proinflammatory-related
chemokines, including RANTES via respective CCRs, has been shown to
possibly involve the trafficking of these cells from peripheral tissue
into inflammatory sites (26, 27). Subsequently, iDCs may
be converted into mDCs by extracellular stimuli, including IL-1,
TNF-, LPS, and CD40 ligation in the inflammation sites, and these
cells may lose their responsiveness to these chemokines (26, 27). In turn, the expression of CCR-7 might be induced in mDCs,
and these cells may acquire responsiveness to MIP-3ß, which is
specifically secreted by TC-rich areas in lymph nodes (26, 27). These properties may account for accumulation of Ag-loaded
mDCs in TC-dependent areas of secondary lymphoid tissue (26, 27). We showed that TGF-ß1 enhanced the RANTES-, MIP-3-,
and SDF-1-mediated chemotaxis via up-regulation of respective
receptors in iDCs and mDCs (Figs. 6 and 7), while the chemotactic
migratory property of mDCs for MIP-3ß was inhibited by TGF-ß1 via
down-regulation of CCR-7 expression (Fig. 9). It has been shown that
TGF-ß1 is produced by various cell types, including TCs,
monocytes/macrophages, and granulocytes, and the production of TGF-ß1
was observed in inflammation sites (44, 45, 46). Collectively,
our findings imply that TGF-ß1 is potentially involved in the
trafficking properties of iDCs as well as mDCs via regulating
chemotaxis to CC and CXC chemokines.
In summary, our findings provide a novel mechanism for the trafficking of human DCs. The chemokines and their respective receptors play important roles in inflammatory and allergic diseases, as well as immune responses (18). In addition, DCs are extensively involved in autoimmune diseases, graft rejection, and viral infections (1, 2, 3, 4). On the other hand, previous studies have shown the induction of MHC products-mediated immune responses after stimulation with tumor Ag-pulsed DCs in clinical trials (47, 48). Recently, the effective targeting of tumor Ag-loading DCs into tumor tissues or lymph nodes around these sites has been shown to be useful for tumor immunotherapy. Defining the precise mechanisms of the chemotactic migratory ability of DCs may provide further insights into the role of these cells in immune-related diseases, and facilitate the use of DCs in vaccinations for cancer treatment.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Tsuneo A. Takahashi, Department of Cell Processing, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. E-mail address:
3 Abbreviations used in this paper: DC, dendritic cell; BM, bone marrow; CXCR, CXC chemokine receptor; FITC-DX, FITC labeled-dextran; HPC, hemopoietic progenitor cell; iDC, immature DC; LY, lucifer yellow; mDC, mature DC; MFI, mean fluorescence intensity; MIP, macrophage-inflammatory protein; SDF, stromal cell-derived factor; TC, T cell.
Received for publication August 23, 1999. Accepted for publication December 13, 1999.
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