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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 34-42
CC Chemokine Receptors, CCR-1 and CCR-3, Are Potentially Involved
in Antigen-Presenting Cell Function of Human Peripheral
Blood Monocyte-Derived Dendritic Cells
By
Katsuaki Sato,
Hiroshi Kawasaki,
Hitomi Nagayama,
Ryo Serizawa,
Junji Ikeda,
Chikao Morimoto,
Kunio Yasunaga,
Noboru Yamaji,
Kenji Tadokoro,
Takeo Juji, and
Tsuneo A. Takahashi
From the Department of Cell Processing, Department of Clinical
Immunology and AIDS Research Center, The Institute of Medical Science,
The University of Tokyo, Tokyo, Japan; the Japanese Red Cross Central
Blood Center, Tokyo, Japan; and the Institute for Drug Discovery
Research, Yamanouchi Pharmaceuticals, Co, Ltd, Tsukuba, Japan.
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ABSTRACT |
We examined the potential involvement of two CC chemokine receptors
(CCRs), CCR-1 and CCR-3, in the functional activation of
granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4)-generated human peripheral blood monocyte-derived immature dendritic cells (DCs). Flow cytometric analysis showed that
CCR-1, CCR-3, CCR-5, and CXC chemokine receptor (CXCR)-4 were expressed
on the cell surface of monocyte-derived DCs. Treatment with a
monoclonal antibody (MoAb) to either CCR-1 or CCR-3 but not MoAbs to
CCR-5 and CXCR-4 abolished chemotactic migration of monocyte-derived
DCs. The DCs treated with either the anti-CCR-1 MoAb or anti-CCR-3
MoAb were less efficient than untreated DCs in proliferation of
allogeneic T cells (TCs) and TC-derived secretion of interferon-
(IFN-). The homotypic aggregation of DCs and heterotypic aggregation
of DCs with TCs were suppressed by the anti-CCR-1 MoAb or anti-CCR-3
MoAb. These results indicate that CCR-1 and CCR-3 specifically regulate
interaction of TCs and DCs in the process of antigen presentation.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
DENDRITIC CELLS (DCs) are unique
professional major antigen (Ag)-presenting cells (APCs) capable of
stimulating resting T cells (TCs) in the primary immune response and
are more potent APCs than peripheral blood monocytes or B
cells.1 DCs are also critically involved in the autoimmune
diseases, graft rejection, and human immunodeficiency virus infection
and the generation of T-cell-dependent antibodies
(Abs).1-4 They capture and process Ag in nonlymphoid
tissues and then migrate to T-cell-dependent areas of secondary
lymphoid organs through afferent lymph or the blood stream to prime
native TCs and initiate the immune response.5 During this
process, DCs lose Ag-capturing/processing ability as they differentiate
into mature, fully stimulatory APCs.6
The 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 Recently, an in vitro culture system
that allows progenitors in peripheral blood, bone marrow, and cord
blood to differentiate into DCs has been established, and it has shown
the basic mechanisms underlying the properties of
DCs.7-13 DCs originate from CD34+
pluripotent hematopoietic progenitor cells in the bone marrow and cord
blood.7,14-17
The chemokines are crucially involved in inflammatory and immunological
responses via their unique capacity to recruit selective leukocyte
subsets.18-21 The chemokines have also been implicated not
only in regulation of normal leukocyte recirculation and homing but
also in certain physiological and pathogenic processes, including hematopoiesis, angiogenesis, allergy, autoimmune diseases, and viral
infectious diseases.22 Chemokines are a group of
approximately 70 to 90 amino acid structually related polypeptides,
most of which contain four conserved cysteine residues in their primary amino acid sequence.23,24 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.23,24
The specific effects of chemokines on the target cell types are
mediated by a family of G-protein-coupled seven-transmembrane receptors.22 Ligand specificities of 12 chemokine receptors have thus far been identified; 4 of the receptors are specific for CXC
chemokines (CXCR1-4),22 6 of them are specific for CC chemokines (CCR1-6),2 and the Duffy antigen receptor (DARC) binds both CC and CXC chemokines.25 In addition, distinct
chemokines appear to act on more than one receptor type in
vitro.22 However, there is increasing evidence to suggest
that this redundancy does not occur in vivo.26
Recent reports have shown that several CC and CXC chemokines receptors
were found to be expressed on some DCs at the transcriptional level,27-29 and these molecules have been implicated to
mediate in part the trafficking of DCs from blood to tissues and then to lymph nodes, where they form a close association with TCs in the
process of Ag presentation. However, the precise roles of these
chemokine receptors in the process of DC-mediated activation of TCs
remain still unknown due to the difficulty in analyzing these phenomena
using available materials such as specific monoclonal antibody (MoAb)
to these molecules.
To better understand how chemokine receptors expressed on the cell
surface of DCs regulate their APC functions, we therefore generated
MoAbs specific to two human CC chemokine receptors, CCR-1 and CCR-3
(Kawasaki et al, manuscript submitted). In this report, we
examined the potential roles of CCR-1 and CCR-3 in the process of
monocyte-derived DC-mediated TC activation. We showed here, in this
respect, that the two CC chemokine receptors, CCR-1 and CCR-3, were
involved in the APC function of immature DCs derived from peripheral
blood monocytes by using exogenous granulocyte-macrophage
colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4). Our
results suggest that the chemokines and their respective receptors play
a crucial role in APC function of DCs.
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MATERIALS AND METHODS |
Media and reagents.
The medium used throughout was RPMI 1640 supplemented with 2 mmol/L
L-glutamine, 50 µg/mL streptomycin, 50 U/mL penicillin, and 10%
heat-inactivated fetal calf serum (FCS). GM-CSF was kindly provided by
Kirin Brewery (Tokyo, Japan). IL-4 and regulated on activation normal
T-cell expressed and secreted (RANTES) were purchased from Pepro Tech
(London, UK). Fluorescein isothiocyanate-labeled dextran (FITC-DX) and
lucifer yellow (LY) were purchased from Molecular Probes, Inc (Eugene,
OR). Phorbol myristate acetate (PMA) and ionomycin (IoM)
were purchased from Sigma (St Louis, MO). The specific MoAbs to CCR-5
and CXCR-4 were purchased from Pharmingen (San Diego, CA).
Preparation of MoAbs to CCR-1 and CCR-3.
cDNA for CCR-1 and CCR-3 were polymerase chain reaction (PCR)-amplified
from cDNA that had been reverse-transcribed from total RNA of human
peripheral blood mononulear cells (PBMCs) stimulated by 5 µg/mL
phytohemagglutinin (Sigma) for 3 days. Amplified cDNA fragments were
subcloned into EF-1a promoter-driven mammalian expression
vector30 and stably expressed in mouse preB-cell lymphoma
B300-19.31 The expression of CCR-1 was flow cytometrically verified by staining with rabbit anti-CCR-1 antiserum kindly provided to us by Dr K. Matsushima (University of Tokyo, Tokyo,
Japan).32 The expression of CCR-3 was confirmed by
ligand-induced calcium influx. Female BALB/c mice were
intraperitoneally immunized with 1 × 107
transfectants 6 times in total over a 3-month period. Three days after
final immunization, mice were killed and spleen cell suspension was
fused with P3XAg8.33 Supernatant of growing
hybridomas was selected for its ability to positively stain
transfectants and negatively stain wild-type B300-19 cell. Anti-CCR-1
clone #141-2 and anti-CCR-3 clone #444-11 were chosen. Both clones
were IgG1 isotype.
In vitro generation and culture of human DCs.
DCs were generated from PBMCs as described previously,8-10
with some modification. Briefly, PBMCs were obtained from 30 mL of
leukocyte-enriched buffy coat from healthy donors by centrifugation with use of Ficoll-Hypaque (Pharmacia Fine Chemicals, Uppsala, Sweden),
and the light-density fraction from the 42.5% to 50% interface was
recovered. The cells were resuspended in culture medium and allowed to
adhere to 6-well plates. After 2 hours at 37°C, nonadherent cells
were removed and adherent cells (~90% CD14+ cells) were
cultured in 3 mL of medium supplemented with GM-CSF (50 ng/mL) and IL-4
(250 ng/mL). After 7 days of culture, DCs were harvested, washed, and
used for subsequent experiments. The resulting cell preparation
contained more than 90% DCs as assessed by morphology and
fluorescence-activated cell sorting (FACS) analysis. The
cell differentiation was monitored by light microscopy.
Isolation of TCs from PBMCs.
Peripheral blood TCs were prepared using a T-cell enrichment
immunocolumn (Biotex Laboratories, Inc, Edmonton, Alberta, Canada) from
leukocyte-enriched buffy coat as described above. T-cell preparations
were typically greater than 90% pure by anti-CD3 MoAb.
Flow cytometry.
For surface marker analysis, DCs were cultured with one of the
following MoAbs conjugated to FITC or phycoerythrin (PE)
for direct fluorescein: CD1a (Coulter Immunology, Hialeah, FL); CD3, CD4, CD11c, CD14, and HLA-DR (all from Becton Dickenson, Mountain View,
CA); and CD40, CD80, CD86, CCR-5, and CXCR-4 (all from Pharmingen). Cells were also stained with the corresponding FITC- or PE-conjugated isotype-matched control MoAb (all from Becton Dickinson). In indirect staining, cells were incubated with the anti-CCR-1 MoAb, anti-CCR-3 MoAb, or biotin-conjugated anti-CXCR-4 MoAb for 30 minutes at 4°C,
washed twice with cold phosphate-buffered saline (PBS), and subsequently stained with FITC-conjugated antimouse IgG (Becton Dickinson) or FITC-conjugated avidin (Becton Dickinson) for 30 minutes
at 4°C. Thereafter, the cells were washed twice and suspended in
PBS containing 0.2 µg/mL propidium iodide (Sigma) to allow exclusion
of dead cells. Analysis of fluorescence staining was performed with a
FACSCalibur flow cytometer (Becton Dickinson) and CELLQuest Software.
Preparation of culture conditioned medium (CM).
Culture CM was prepared as follows. TC-conditioned media were obtained
from culture of purified TCs (5 × 107) unstimulated
or stimulated with PMA (50 ng/mL) plus IoM (500 ng/mL) in 5 mL of
serum-free medium for 24 hours at 37°C. TCs/DCs-conditioned medium
was prepared from coculture of TCs (5 × 107) and DCs
(5 × 106) in 5 mL of serum-free medium for 5 days at
37°C. DCs-conditioned medium was obtained from culture of DCs
(107) in 5 mL of serum-free medium for 3 days at 37°C.
Each supernatant was collected, and cell-free supernatants were
obtained after centrifugation at 800g for 5 minutes and passage
through a 0.22-µm filter (Milipore Corp, Bedford, MA) and then stored
at  20°C before use.
Assay for chemotaxis.
The in vitro migration of cells prepared as described above in response
to RANTES (1 µg/mL) or CMs prepared as described above was assessed
in a Transwell cell culture chamber (Costar 3422; Costar, Cambridge,
MA) as described previously,28 with some modification. In
brief, polycarbonate filters with 8.0-µm pore size (Nucleopore,
Pleasanton, CA) were precoated with 5 µg of gelatin 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 various concentrations from 0.1 to 10 µg/mL of the MoAbs to CCR-1, CCR-3, CCR-5, CXCR-4, or control IgG
(cont. IgG; Sigma) for 30 minutes at 37°C, and 100 µL of the cell
suspension (106) was added to the upper compartment of the
chamber. RANTES or CM diluted in serum-free culture medium was loaded
in the lower compartment. After 2 hours of incubation, the filters were
fixed with methanol and stained with hematoxylin and eosin. 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
×400, and each assay was performed in triplicate. The data were
expressed as the number of migrated cells per field.
Endocytosis assay with FITC-DX and LY.
The methods used to determine the endocytotic activity of in
vitro-generated DCs have previously been described.10
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 minutes at 37°C. After incubation, cells were washed four times with ice-cold PBS and
analyzed by flow cytometry as described above.
Mixed leukocytes reaction (MLR) and detection of
interferon- (IFN-) by enzyme-linked
immunosorbent assay (ELISA).
Responding TCs (105) from an unrelated individual
(allogeneic MLR) in the presence or absence of various concentrations
from 0.1 to 10 µg/mL of anti-CCR-1 MoAb, anti-CCR-3 MoAb, or cont. IgG were cultured in 96-well flat-bottom microplates (Costar) with
different numbers of monocyte-derived DCs. Thymidine incorporation was
measured on day 5 by an 18-hour pulse with 0.5 µCi/well of [3H]thymidine (1 µCi/well; specific activity, 5 Ci/mmol; Amersham Life Science, Buckinghamshire, UK). The culture in
each well was monitored by light microscopy. In another experiment, the
culture supernatant of each well was collected, and assay for IFN-
production was performed. IFN- was detected in the
supernatants using a two-site sandwich ELISA (Endogen, Woburn, MA).
Samples were analyzed in serial twofold dilutions in duplicate; the
sensitivity of the assay was 2 pg/mL.
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RESULTS |
Cell surface expression of CCR-1 and CCR-3 in monocyte-derived DCs.
The regulated interactions of TC-derived chemokines with their
respective receptors expressed on DCs are thought to mediate the
controlled recruitment of DCs from resident sites in nonlymphoid organs
into the lymph nodes and spleen to initiate the primary T-cell-dependent immune response of DCs. However, little is known about the involvement of chemokine receptors expressed on DCs in their
APC functions. In an attempt to clarify the role of chemokine receptors
underlying APC functions of human peripheral blood immature DCs, we
generared MoAbs to both human CCR-1 and CCR-3 (Kawasaki et al,
manuscript submitted). To investigate specific
recognition of MoAbs to CCR-1 and CCR-3 for the respective CCRs, the
cell surface expression levels of CCR and CXCR in the transfectants expressing respective CCR-1 or CCR-3 were examined.
Figure 1A shows that transfectants
expressing CCR-1 or CCR-3 exclusively expressed respective CCR. These
results indicate that MoAbs to CCR-1 and CCR-3 specifically recognize
the respective CCRs.
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| Fig 1.
Cell surface expressions of chemokine receptors in
monocyte-derived DCs. (A) Chemokine receptors expression levels in the
transfectants expressing CCR-1 or CCR-3. The transfectants were stained
with anti-CCR-1 MoAbs, anti-CCR-3 MoAbs, biotin-conjugated
anti-CXCR-4 MoAbs, or FITC-conjugated anti-CCR-5 MoAb (thick lines)
or FITC-conjugated mouse Ig (thin lines) for 30 minutes at 4°C. In
an indirect staining, the cells were subsequently stained with
FITC-conjugated antimouse IgG or FITC-conjugated avidin for 30 minutes
at 4°C. (B) Expression levels of chemokine receptors in
monocyte-derived DCs. Monocyte-derived DCs were stained with MoAbs to
the respective chemokine receptors as described above. The cell surface
expression was analyzed by flow cytometry. The results are
representative of three experiments performed with similar results.
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To examine the mechanism by which CCR-1 and CCR-3 exerted in DCs, we
established immature DCs from human peripheral blood monocytes with the
use of GM-CSF (50 ng/mL) plus IL-4 (250 ng/mL). These cells have a
typical dendritic morphology, a phenotype (CD1a+,
CD4+, CD11c+, CD14 ,
CD40+, CD80+, CD86+, and
HLA-DR+), a high pinocytic capacity for FITC-DX or LY, and
high capacity to cause allogeneic TCs to proliferare (data not shown).
Recent studies have shown that DCs express the transcripts for several chemokine receptors27-29; however, their cell surface
expression levels remain still unknown. Therefore, we first examined
the cell surface expressions of CCR-1 and CCR-3, CCR-5, and CXCR-4 in
monocyte-derived DCs with the use of MoAbs to respective chemokine receptors. Flow cytometric analysis showed that monocyte-derived DCs
exhibited various expression levels of both CCR-1 and CCR-3 (Fig 1B).
On the other hand, we observed that another CC chemokine receptor,
CCR-5, was expressed at a low level on monocyte-derived DCs and that
these cells also expressed CXCR-4 (Fig 1B).
Previous studies have shown that CCR-1 is predominantly expressed on
peripheral blood TCs,32 whereas CCR-3 was found to be
expressed only on T-helper 2 (Th2) cells.34 We observed
that the MoAb to CCR-1 stained a large population of peripheral blood TCs, whereas the MoAb to CCR-3 weakly reacted with the bulk population and strongly stained only a small proportion (<5%) of peripheral blood TCs (data not shown).
MoAbs to CCR-1 and CCR-3 suppress DC chemotactic migration.
The trafficking of DCs from blood to tissues and then to lymph nodes
has been thought to be mediated in part by members of the chemokine
superfamily.28 To assess whether CCR-1 and CCR-3 were
involved in trafficking properties of monocyte-derived DCs, the effect
of MoAb to CCR-1 and CCR-3 on the ability of monocyte-derived DCs to
migrate in response to stimuli by various chemoattractants was examined
using a Transwell cell culture chamber. Previous reports have
demonstrated that RANTES, a member of the CC chemokine family, acts as
a potent and selective chemoattractant for monocytes, memory T cells,
natural killer cells, eosinophils, and DCs and possesses binding
affinity and fidelity to CCR-1 and CCR-3.22 To assess
whether monocyte-derived DCs migrated to RANTES via CCR-1
and/or CCR-3, we examined the effects of MoAbs to CCR-1 and
CCR-3 on the migratory capacity of monocyte-derived DCs in response to
RANTES. Figure 2A shows that both the
anti-CCR-1 MoAb and anti-CCR-3 MoAb inhibited chemotactic migration
of monocyte-derived DCs to RANTES in a dose-dependent manner. On the
other hand, treatment of peripheral blood TCs with either of these
MoAbs had a suppressive effect on their migratory capacity to RANTES,
although these inhibitory effects were not significant (data not
shown). To address the role of other chemokine receptors in chemotactic
migratory capacities of monocyte-derived DCs, we examined the effects
of MoAbs to CCR-5 and CXCR-4 on chemotactic migration of
monocyte-derived DCs to RANTES, because CCR-5 also exhibited binding
affinity and fidelity to RANTES, whereas CXCR-4 acts as a receptor for
stromal cell-derived factor-1.22 The treatment of
monocyte-derived DCs with anti-CCR-5 MoAb slightly inhibited the
chemotactic migration to RANTES, whereas anti-CXCR-4 MoAb had little
or no suppressive effect on the chemotactic migratory capacities of
these cells (Table 1). We further examined the combinations of MoAbs to CCR-1, CCR-3, and CCR-5 on the migratory capacity of monocyte-derived DCs to RANTES. The combinations of these
two or three MoAbs to CC chemokine receptors exhibited greater inhibition on the migratory capacity of monocyte-derived DCs to RANTES
than those of each MoAb to CCRs (Table 1). These results indicate that
CCR-1, CCR-3, and CCR-5 contribute to the migratory capacity of
monocyte-derived DCs to RANTES.
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| Fig 2.
CCR-1 and CCR-3 regulate the migratory capacity of
monocyte-derived DCs. Monocyte-derived DCs (106) were
pretreated with the indicated concentrations of anti-CCR-1 MoAb,
anti-CCR-3 MoAb, or cont. IgG for 30 minutes at 37°C and seeded on
the filters precoated on the lower surface with 5 µg of gelatin. (A)
RANTES (1 µg/mL), (B) CM derived from unstimulated or PMA (50 ng/mL)
plus IoM (500 ng/mL)-stimulated TCs, (C) CM derived from coculture of
TCs and DCs, and (D) CM derived from culture of DCs used as a
chemoattractant were added to the lower chamber. After 2 hours of
incubation, the cells that migrated to the lower surface were visually
counted.
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The recruitment of DCs from blood to tissue and then to lymph nodes
where DCs present antigenic materials to TCs is believed to be mediated
in part by identified and/or unknown TC-derived chemokines. We
investigated involvement of CCR-1 and CCR-3 in the chemotactic
migration of monocyte-derived DCs to CM derived from peripheral blood
TC (Fig 2B). CM of PMA/IoM-stimulated peripheral blood TCs induced a
migration of monocyte-derived DCs, although the cells showed little or
no migratory capacity to CM of unstimulated TCs. Treatment of
monocyte-derived DCs with MoAbs to CCR-1 and CCR-3 suppressed
chemotactic migration to CM of PMA/IoM-stimulated TCs. These results
indicate that CCR-1 and CCR-3 are involved in trafficking of
monocyte-derived DCs to TC-derived chemokines.
CCR-1 and CCR-3 regulate DC-mediated triggering activation of TCs.
The interaction of DCs with primed TCs via chemokines and their
respective receptors as well as adhesion/costimulatory molecules is
believed to induce TC activation via APC function. To investigate the
role of chemotactic migration between TCs and DCs, we examined the
effects of MoAbs to CCR-1 and CCR-3 on the capacity of allogeneic TCs
to stimulate monocyte-derived DCs. TCs (105) were
cocultured with various numbers of monocyte-derived DCs (102 to 5 × 104) in the presence or
absence of indicated concentrations of the anti-CCR-1 MoAb,
anti-CCR-3 MoAb, or cont. IgG ranging from 0.1 to 10 µg/mL, and the
proliferative responses of TCs were measured on day 5. MoAbs to CCR-1
and CCR-3 significantly abolished the allostimulatory potential of
monocyte-derived DCs (Fig 3A and B), and
these results were concomitant with the capacity of DCs to stimulate
TC-derived IFN- production (Fig 4A and
B). We also observed that these MoAbs had little or no effect on cell
surface expression levels of CD1a, CD11c, CD40, CD80, CD86, and HLA-DR in these monocyte-derived DCs (data not shown). These results indicate
that CCR-1 and CCR-3 expressed on TCs and DCs are potentially involved
in DC-mediated TC activation.
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| Fig 3.
MoAbs to CCR-1 and CCR-3 suppress capacity of
monocyte-derived DCs to stimulate allogeneic TCs proliferation. TCs
purified from PBMCs (105) were cultured (A) with
monocyte-derived DCs (104) in the presence of the indicated
concentrations of anti-CCR-1 MoAb, anti-CCR-3 MoAb, or cont. IgG or
(B) with different numbers of monocyte-derived DCs in the presence of
10 µg/mL anti-CCR-1 MoAb, anti-CCR-3 MoAb, or cont. IgG. The
proliferative response was measured on day 5. Values are the mean ± SD obtained for triplicate cultures and are representative of those
obtained in two individual experiments.
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| Fig 4.
MoAbs to CCR-1 and CCR-3 suppress capacity of
monocyte-derived DCs to stimulate allogeneic TCs-derived IFN-
secretion. TCs purified from PBMCs (105) were cultured (A)
with monocyte-derived DCs (104) in the presence of the
indicated concentrations of anti-CCR-1 MoAb, anti-CCR-3 MoAb, or
cont. IgG or (B) with different numbers of monocyte-derived DCs in the
presence of 10 µg/mL anti-CCR-1 MoAb, anti-CCR-3 MoAb, or cont.
IgG. The IFN- secretion in the culture supernatants was measured by
ELISA on day 5. Values are the mean ± SD obtained for triplicate
cultures and are representative of those obtained in two individual
experiments.
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CCR-1 and CCR-3 are directly involved in interaction of
monocyte-derived DCs with TCs.
To address role of chemokines and their receptors in DC aggregations,
we cultured monocyte-derived DCs with allogeneic TCs in the presence or
absence of the anti-CCR-1 MoAb, anti-CCR-3 MoAb, or cont. IgG
(Fig 5). Using light microscopy, treatment of the cells with these MoAbs was observed to result in suppression of
their homotypic aggregation. To further examine the consequences of
inhibiting the homotypic aggregation of DCs via CCR-1 and CCR-3, we
evaluated the effects of MoAbs to CCR-1 and CCR-3 on the migratory capacity of monocyte-derived DCs to CM derived from DC cultures. Figure
2C shows that CM of DCs induced a chemotactic migration of
monocyte-derived DCs. On the other hand, treatment of monocyte-derived DCs with MoAbs to CCR-1 and CCR-3 inhibited their migratory capacities. These results indicate that interaction of chemokines derived from DCs
with CCR-1 and CCR-3 expressed on DCs in part contributes to DC
aggregations.
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| Fig 5.
CCR-1 and CCR-3 are involved in clustering of
monocyte-derived DCs with TCs. TCs purified from PBMCs
(105) were cultured with monocyte-derived DCs
(104) in the absence (A) or presence of 10 µg/mL cont.
IgG (B), anti-CCR-1 MoAb (C), or anti-CCR-3 MoAb (D). The coculture
of monocyte-derived DCs with TCs was measured on day 5. Original
magnification × 200. The results are representative of two
experiments performed with similar results.
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The priming of TC activation by DCs is thought to be mediated by
clustering of DCs and TCs. We further evaluated the effect of MoAbs to
CCR-1 and CCR-3 on cluster formation between TCs and DCs. As shown in
Fig 5, coculture of monocyte-derived DCs with TCs in the presence of
the anti-CCR-1 MoAb and anti-CCR-3 MoAb, but not cont. IgG, resulted
in heterotypic aggregation of monocyte-derived DCs with TCs. To clarify
the CCR-1- and CCR-3-mediated mechanisms underlying formation of
TC-DC aggregation, we performed a chemotactic migration assay of
monocyte-derived DCs to CM derived from coculture of monocyte-derived
DCs with TCs (Fig 2D). We found that CM derived from coculture of
monocyte-derived DCs with TCs promoted the migration of DCs, and MoAbs
to CCR-1 and CCR-3 significantly inhibited their mobility in a
dose-dependent manner. These results indicate that CCR-1 and CCR-3 are
critically involved in APC function of monocyte-derived DCs.
| |
DISCUSSION |
Trafficking of DCs from local nonlymphoid areas to lymphoid tissue is
believed to be a crucial event in the process of presentation of
antigenic materials to naive or memory TCs. However, little is known
about the precise role of chemotactic responses in APC functions of
DCs. To clarify the importance of chemotactic events in interaction of
DCs with TCs, we generated MoAbs to CCR-1 and CCR-3 (Kawasaki et al,
manuscript submitted). The present study has demonstrated
that monocyte-derived DCs expressed CCR-1 and CCR-3 as well as CCR-5
and CXCR-4 on the cell surface, treatment of DCs with anti-CCR-1 MoAb
or anti-CCR-3 MoAb, but not anti-CCR-5 MoAb or anti-CXCR-4 MoAb,
markedly inhibited allogeneic T-cell responses, including proliferation
and IFN- secretion via suppression of chemotactic migration between
TCs and DCs.
A series of recent studies showed that several CCRs or CXCRs were
constitutively expressed on a group of DCs at the transcriptional level.27-29 To the best of our knowledge, we are the first
to have detected CCR-1, CCR-3, CCR-5, and CXCR-4 on the cell surface of monocyte-derived DCs by flow cytometric analysis using MoAbs to respective CCRs or CXCR (Fig 1). On the other hand, peripheral blood
TCs also constitutively expressed CCR-1, although the cells possessed
relatively little expression levels of CCR-3 (data not shown). A
previous report showed that CCR-1 was expressed on the surface of
peripheral blood TCs,32 whereas CCR-3 was selectively expressed on the subset of Th2 cells.35 These data suggest
that CCR-1 and CCR-3 expressed on subset of TCs and DCs may be involved in Ag presentation.
Previous studies have shown that subsets of DCs migrate in response to
various sets of chemokines.28,35-37 We showed that monocyte-derived DCs exhibited a migratory response to RANTES and MoAbs
to CCR-1 or CCR-3 significantly suppressed it, although not completely
(Fig 2A). Furthermore, MoAb to CCR-5 slightly inhibited chemotactic
migratory actions of monocyte-derived DCs, whereas MoAb to CXCR-4 did
not suppress it (Table 1). These results indicate that monocyte-derived
DCs migrated to RANTES via CCR-1, CCR-3, and CCR-5. We also showed that
the combinations of these two or three MoAbs to CC chemokine receptors
did not completely suppress the RANTES-mediated chemotactic migration
of monocyte-derived DCs, although these combinations elicited greater
inhibition on it than those of each MoAb. These results imply that
other chemokine receptor(s) may also act as a receptor for RANTES on
monocyte-derived DCs.
We also observed that these MoAbs inhibited chemotactic migration of
monocyte-derived DCs to CM-derived PMA plus IoM-stimulated TCs (Fig
2B). These results imply that chemokines secreted by TCs and their
respective receptors, including CCR-1 and CCR-3 expressed on DCs, may
directly regulate chemotactic migration of DCs to TCs.
Recent studies have shown that TCs and DCs can secrete various sets of
CXC- or CC-chemokines.28,35-37 We observed that CM-derived coculture of TCs and DCs caused DCs to migrate, and both the
anti-CCR-1 MoAb and anti-CCR-3 MoAb suppressed these migratory events
(Fig 2C). These results suggest that chemokines produced by TCs and DCs
and CCR-1 and CCR-3 expressed on these cells may contribute to
chemotactic migratory events in the process of Ag presentation.
DCs found in TC-dependent areas have been implicated to interact with
TCs via Ag presentation, followed by activation of TCs to
proliferate in secondary lymphoid tissue. We showed that MoAbs to CCR-1
or CCR-3 significantly suppressed the capacity of DCs to stimulate
proliferation of allogeneic TCs and producion of IFN- (Figs 3 and
4). These phenomena led us to hypothesize that CCR-1 and CCR-3 were
crucially involved in the capacity of monocyte-derived DCs to activate
TCs. Indeed, we observed that heterotypic aggregation of
monocyte-derived Dcs with TCs was mostly inhibited by MoAbs to CCR-1 or
CCR-3 (Fig 5). Our results imply that suppression of chemotaxis of DCs
and TCs by the anti-CCR-1 MoAb or anti-CCR-3 MoAb may subsequently
lead to eliminate specific interaction of TCs and DCs, resulting in
inhibition of activation of TCs.
In summary, our results indicate that MoAbs to CCR-1 or CCR-3
negatively regulated activation of TCs by monocyte-derived DCs via
suppression of their migratory capacity. The capacity of MoAbs to CCR-1
or CCR-3 to inhibit functions of monocyte-derived DCs was markedly, but
not completely, suppressed, indicating that other CC or CXC chemokine
receptors may also be involved in the functions of DCs. To further
characterize the roles of other CC or CXC chemokine receptors, MoAbs to
these molecules have been prepared and are now under investigation in
our laboratories. The chemokines and their respective receptors play
important roles in inflammatory and allergic diseases as well as immune
responses. On the other hand, DCs are critically involved in autoimmune
diseases, graft rejection, and virus infection.1-4 Thus,
the availability of MoAbs to CCR-1 or CCR-3 may be potentially useful
for prevention of immune-related diseases.
| |
ACKNOWLEDGMENT |
The authors thank H. Takahashi for excellent assistance.
| |
FOOTNOTES |
Submitted February 19, 1998;
accepted July 22, 1998.
K.S. and H.K. contributed equally to this study.
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 Tsuneo A. Takahashi, DSc, Department of
Cell Processing, The Institute of Medical Science, The University of
Tokyo, Tokyo, Japan; e-mail: takahasi{at}ims.u.-tokyo.ac.jp.
| |
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Abstract
Introduction