By
From the * Geneva Biomedical Research Institute, GlaxoWellcome Research and Development,
Geneva, Switzerland; University of British Columbia, Vancouver, Canada; and § Istituto di Ricerche
Farmacologiche Mario Negri, Milan, Italy
Dendritic cells are potent antigen-presenting cells involved in the initiation of immune responses. The trafficking of these cells to tissues and lymph nodes is mediated by members of the chemokine family. Recently, a novel CC chemokine known as MIP-3 or liver and activation-regulated chemokine has been identified from the EMBL/GenBank/DDBJ expressed sequence tag database. In the present study, we have shown that the messenger RNA for MIP-3
is expressed predominantly in inflamed and mucosal tissues. MIP-3
produced either synthetically or by human embryonic kidney 293 cells is chemotactic for CD34+-derived dendritic
cells and T cells, but is inactive on monocytes and neutrophils. MIP-3
was unable to displace
the binding of specific CC or CXC chemokines to stable cell lines expressing their respective
high affinity receptors, namely CCR1-5 and CXCR1 and CXCR2, suggesting that MIP-3
acts through a novel CC chemokine receptor. Therefore, we used degenerate oligonucleotide-based reverse transcriptase PCR to identify candidate MIP-3
receptors in lung dendritic cells.
Our results show that the orphan receptor known as GCY-4, CKRL-3, or STRL-22 is a specific receptor for MIP-3
, and that its activation leads to pertussis toxin-sensitive and phospholipase C-dependent intracellular Ca2+ mobilization when it is expressed in HEK 293 cells.
Dendritic cells (DCs)1 play an essential role in the induction of immune responses (1). DC precursors are
thought to originate in the bone marrow and subsequently
migrate into the peripheral tissues and lymph nodes. The
main function of these cells is to sample and process incoming antigenic material, which is then presented to naive T
cells, either locally or within regional lymph nodes, resulting in T cell activation and the generation of immune responses necessary to clear the infection. The trafficking of
DCs from blood to tissues and then to lymph nodes is mediated in part by members of the chemokine superfamily.
Chemokines are small molecular weight (8-10-kD) proteins, most of which contain four conserved cysteine residues in their primary amino acid sequence. There are two
major groups: the CXC chemokines in which the two
NH2-terminal cysteines are separated by a single amino
acid, or the CC chemokines in which the two NH2-terminal cysteines are adjacent (2). A third type of chemokine represented by lymphotactin, contains only two of the four
conserved cysteines (3). Recently, the prototype of a fourth
class of chemokines has been described in which the first
two of the four conserved cysteines are separated by three
amino acids (CX3C) (4).
Chemokines were originally identified through their
chemoattractant effects on different leukocyte populations,
but other approaches have included the selective cloning of
secreted proteins using the signal sequence trap (5, 6) or by
subtractive cloning methods (7). In addition to this, a number of putative chemokine sequences have recently been
reported in expressed sequence tag (EST) databases (8).
About 30 chemokines are known to date and this number
is likely to increase as more sequence information is obtained from genome sequencing projects.
The specific effects of chemokines on target cell types
are mediated by a family of G protein-coupled seven-transmembrane receptors. 10 human chemokine receptors have
been identified so far; 4 of which are specific for CXC
chemokines (CXCR1-4) (9); 5 of which are specific
for CC chemokines (CCR1-5) (13); and the Duffy antigen receptor (DARC), which binds both CC and CXC chemokines (18). All of these receptors bind more than one
chemokine at high affinity with the exception of CXCR1,
which is specific for IL-8, and CXCR4, which so far appears to be specific for stromal cell-derived factor-1 (SDF-1).
In addition, distinct chemokines appear to act on more
than one receptor type. However, there is increasing evidence to suggest that this redundancy does not occur in
vivo (19). Despite the overlapping ligand specificities shown by chemokine receptors, the number of chemokines identified far exceeds the number of known receptors. Degenerate oligonucleotide-based PCR cloning strategies used to
identify chemokine receptor cDNAs have also resulted in
the cloning of numerous orphan receptors (20). Although
these orphan receptors show significant levels of identity
(30-50%) at the amino acid level to the known chemokine receptors, their natural ligands remain to be identified. It is more than likely that the ligands will turn out to be many
of the newly identified EST-derived chemokines, but
progress in this area is slow due to the limited availability of
these proteins for testing.
To appreciate fully the role of chemokines in diverse
biological processes and the mechanisms by which they
control the recruitment of immune cells necessitates the
identification and characterization of their specific receptors. We and others have recently identified a novel CC
chemokine from the EMBL/GenBank/DDBJ EST database known as MIP-3 Materials.
Restriction enzymes and DNA-modifying enzymes
were purchased from New England Biolabs (Beverly, MA) unless
otherwise stated. AmplitTaqTM was from Perkin-Elmer Cetus
(Norwalk, CT). AMV reverse transcriptase was from Promega
(Madison, WI). All cell culture reagents were from GIBCO BRL
Life Sciences (Paisley, UK). MIP-1 Purification of Leukocyte Populations.
Leukocyte populations used
for chemotaxis assays or for RT-PCR experiments were purified
from the buffy coats of normal healthy blood donors after centrifugation through Ficoll-Paque (Pharmacia Biotech, Uppsala,
Sweden). The PBMCs were subfractionated into T cells and
monocytes after rossetting by sheep RBCs. CD4 and CD8 T cell
subpopulations were further purified using T cell purification columns (R&D Systems). NK cell populations were purified as described previously (29). Eosinophils were purified from the fresh
blood of asymptomatic hypereosinophilic donors by negative selection from the granulocyte pellet using anti-CD16 monoclonal
antibodies (30). The purity of each cell population was determined by FACS®, and only populations showing >99% purity
were used for experiments, except for monocytes that were routinely >80% pure. Peripheral blood monocyte-derived DCs
were prepared as described previously (31). CD34+-derived DCs
were obtained by CD34+ selection from PBMCs using CD34+
columns (Minimacs, Milteny Biotec, Bergisch Gladbach, Germany) as described (32). In brief, CD34+ cells were cultured at
5 × 104 cells/ml in RPMI-1640 containing 10% FCS supplemented with growth factors. During the first week, the following
factors were used: stem cell factor (50 ng/ml) (Amgen, Thousand
Oaks, CA) and GM-CSF (50 ng/ml) (Sandoz, Basel, Switzerland). During the second week, cells were maintained in GM-CSF and TNF (10 ng/ml) (Basf Knoll, Ludwigschaffen, Germany). Cells (70-80% CD1a+, MHC class II+, and CD80+) were
used after 12-15 d of culture. Lung macrophages and lung DCs
were purified from resected human lung using the method of Aubry et al. (33). In brief, lung tissue was dissociated with DNAse I and collagenase type I (Sigma Chemical Co., St. Louis, MO). The cell suspension was subjected to centrifugation
through Ficoll and the resultant mononuclear cell layer was subsequently washed twice and plated at a concentration of 2 × 106
cells/ml on 10-cm-diam petri dishes. After incubation at 37°C for
1 h, nonadherent cells were removed by washing with HBSS. Cells adhering to the plastic were incubated overnight at 37°C. Loosely adherent mononuclear cells (LAMCs) were recovered by
washing the monolayers four times with HBSS. LAMCs were
then sorted by FACS®. Macrophages were sorted based on their
autofluorescence. The remaining population was then sorted using four-color fluorescence, selecting for the CD40+, CD3 Preparation of Total RNA and RT-PCR.
Total RNA was prepared from leukocytes using the TrizolTM reagent (Life Sciences,
Paisley, Scotland) according to the instructions of the manufacturer. Reverse transcriptase reactions were performed on 3 µg of
total RNA using an oligo dT primer with a reverse transcription kit (Promega) in a final volume of 20 µl. 2-µl aliquots of each reverse transcriptase reaction were then subjected to 40 cycles of
PCR in a 50 µl reaction mixture containing 10 mM Tris-HCl
buffer, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPS,
2.5 U of AmpliTaq, and 1 µM each of forward and reverse
primer in a Crocodile III thermal cycler (Appligene, Strasbourg,
France) or a Perkin-Elmer DNA thermal cycler. As control, PCR
reactions were performed on RNA samples that had been incubated in the absence of reverse transcriptase. As a check for the
quality of the cDNA used in each PCR reaction, we used glyceraldehyde 3-phosphate dehydrogenase primers as described previously (16). The identity of PCR products that corresponded to
the predicted size was verified by sequencing, using the same
primers as for the PCR reaction, after gel purification using a
Wizard PCR preps kit (Promega).
Cloning of MIP-3 Analysis of MIP-3 Degenerate Oligonucleotide PCR to Identify Chemokine Receptors
Expressed in Human Lung DCs.
Total RNA was isolated from
5 × 105 FACS® purified human lung DCs using TrizolTM. Single-stranded cDNA was prepared from the resultant RNA using
the Promega reverse transcription system. One-tenth of the reaction mixture was subjected to 40 cycles of PCR (95°C for 1 min,
37°C for 1 min, and 72°C for 1 min) using 3 µM of each degenerate oligonucleotide primer (5 Cloning of Full-Length DCCR2.
The full coding sequence of
DC chemokine receptor 2 (DCCR2) was cloned from human
lung DCs by RT-PCR essentially as described above using 1 µM
of specific primers (based on the sequence of full-length cDNA
deposited in the EMBL/Genbank/DDBJ database under accession number U45984) (5 Transient Expression of DCCR2 in HEK 293 Cells.
HEK 293 cells were maintained in DMEM-F12 medium containing 10%
heat-inactivated FCS, 2 mM glutamine, and 100 U/ml penicillin-streptomycin. 24 h before transfection cells were plated at 2 × 106 cells per 10-cm-diam petri dish or at 105 cells per well in 96-well black-walled microtiter plates (Polyfiltronics Group, Inc.,
Rockland, MA) precoated with 0.1% gelatin and 10 µg/ml poly
L-lysine (Sigma Chemical Co.). Cells were transfected using a calcium phosphate transfection kit (Life Sciences) according to the
instructions of the manufacturer. Cytosolic free calcium concentration ([Ca2+]i) and radioligand binding were measured 36-72 h
after transfection.
Transient Expression of MIP-3 or liver and activation-regulated
chemokine (LARC) (21, 22). Here, we show that synthetic
or recombinantly expressed MIP-3
is a potent chemoattractant for certain DCs and T cells. MIP-3
was unable to
bind to any of the known CC chemokine receptors (CCR1-5) or to CXCR1 or CXCR2, suggesting the existence of a specific receptor for this chemokine. Therefore,
we used degenerate oligonucleotide-based RT-PCR to identify candidate receptors in cells that responded to MIP-3
.
As chemokine receptor activation typically leads to pertussis toxin-sensitive phospholipase C activation resulting in
calcium mobilization in most systems (23), we used a fluorescent imaging plate reader to measure changes in intracellular cytosolic free calcium concentration ([Ca2+]i) in human
embryonic kidney (HEK) 293 cells transiently expressing high levels of orphan chemokine-like receptors to identify
the putative MIP-3
receptor. Here, we show that an orphan receptor, formerly known as GCY4, STRL-22 (24),
or CKR-L3 (25), is a specific receptor for the novel CC
chemokine, MIP-3
, and its activation by MIP-3
leads to
both calcium influx and mobilization in cells in which it is
expressed.
, RANTES, and IL-8 were
expressed in Escherichia coli and prepared as described earlier (26,
27). MCP-1 was obtained using similar methodology. SDF-1
was prepared at Glaxo, Inc. (Research Triangle Park, NC). Synthetic MIP-3
, MIP-5, and the NH2-terminal chemokine domain of the CX3C chemokine also known as fractalkine (4) were
chemically synthesized according to published methods (28) using
the sequences discussed in Wells and Peitsch (8). All other recombinant chemokines used in this study were purchased from
R&D Systems, Inc. (Minneapolis, MN) or Peprotech, Inc.
(Rocky Hill, NJ). Radiolabeled [125I] MIP-3
was prepared by
Amersham (Cardiff, UK) to a specific activity of 2,000 Ci/mmol.
Fluo-3 AM was purchased from Molecular Probes, Inc. (Eugene,
OR). Oligonucleotide primers were synthesized by Microsynth
(Balgach, Switzerland).
,
CD14
, CD20
, and CD56
population representing lung DCs.
The lung DCs (>95% purity) express high levels of HLA class II,
CD80, and CD40 and are able to induce a strong mixed lymphocyte reaction.
.
The full coding sequence of MIP-3
was
cloned by RT-PCR using specific primers (5
forward CGG
GAT CCA CCA TGT GCT GTA CCA AGA GTT TG) and
(5
reverse CGG AAT TCC AGT TTT TAC ATG TTC TTG AC) based on the sequence deposited in the EMBL/GenBank/
DDBJ EST database (accession number D31065) for 40 cycles of
PCR (95°C for 1 min, 37°C for 1 min, and 72°C for 1 min). The
cDNA template used in the PCR reaction was either 2 µl of
16 different Quick Clone cDNAs (Clontech, Palo Alto, CA) or
was generated by reverse transcription of isolated leukocyte
RNAs (as described above). PCR products corresponding to the
predicted size of 306 bp were gel purified and subcloned as
BamHI-EcoRI fragments into the mammalian cell expression
vector pcDNA3.1(+) (Invitrogen, San Diego, CA) and sequenced using T7 and pcDNA3AS primers. Two of the resultant
clones, MIP-3
-11, which contained the full error-free coding
sequence of MIP-3
, and MIP-3
-16, which contained a 3-bp deletion in the putative signal peptide coding sequence, were used in subsequent experiments.
Messenger RNA Expression.
Multiple tissue
Northern blots (Clontech) and Northern Territory blots (Invitrogen) were probed with a 306-bp BamHI-EcoRI insert from
MIP-3
-11 according to the instructions of the manufacturer. [
-32P]dCTP-labeled probes were generated using an Amersham
Rediprime kit.
forward GAY MGI TAY YTI
GCI ATH GTX CA and 5
reverse RMR TAI ADI AII GGR TTI AXR CA ) in a Perkin-Elmer DNA thermal cycler. PCR
reaction products were visualized on 1% agarose gels containing
0.5 µg/ml ethidium bromide. Reaction products migrating at the
predicted size (500-550 bp) were gel purified, subcloned into
pBluescript II KS(
) and sequenced on an Applied Biosystems
ABI 377 machine using T3 and T7 primers as described elsewhere (16).
forward TCA ATG AAT TTC AGC
GAT GTT TTC G) and (5
reverse CTA TCA CAT AGT GAA
GGA CGA CGC) using 40 cycles of PCR (95°C for 2 min, 55°C
for 2 min, and 72°C for 2 min). PCR products corresponding
to the predicted size of 1.1 kb were gel purified and subcloned
into the EcoRV site of the mammalian cell expression vector
pcDNA3.1(+) and sequenced using T7 and pcDNA3AS primers.
One of the resultant clones, DCCR2-10, which was identical to
the U45984 coding sequence (also known as GCY-4), was subsequently used for expression studies.
by HEK 293 Cells.
HEK 293 cells plated in 10-cm diameter petri dishes were transfected as described above with pcDNA3.1(+) MIP-3
or pcDNA3.1(+) chloramphenicol acetyl transferase (CAT) as control except that calcium phosphate precipitate containing medium was replaced
with serum-free medium 16 h after transfection. Conditioned
medium was collected 48 h later. Cell debris was removed by
centrifugation at 2,000 rpm for 5 min and diluted medium was
used directly in chemotaxis assays.
, conditioned medium was concentrated 10-fold
using Centriprep-3 ultrafiltration units (Millipore Corp., Milford, MA) and subjected to reverse phase HPLC using an Aquapore
RP-300 7 µm column (0.2 × 22 cm) equilibrated in 0.1% trifluoroacetic acid. Proteins were eluted with a linear gradient of
22.5-45% acetonitrile in 0.1% trifluoroacetic acid and analyzed
by 4-20% SDS-PAGE. Bands migrating at the predicted molecular mass for chemokines (8 kD) were purified and sequenced on a
(494 Procise; Applied Biosystems) protein sequencer.
Calcium Fluorometry.
Cytosolic-free calcium determinations
were conducted in monolayers of HEK 293 cells transiently expressing the DCCR2 receptor by adapting the method of Capponi et al. (34). In brief, black-walled 96-well microtiter plates
containing confluent monolayers of transfected cells were washed
with 150 µl/well serum-free DMEM-F12 medium containing
2 mM glutamine and 100 U/ml penicillin-streptomycin (serum-free medium). The washes were removed, and the monolayers were incubated at 37°C in a water-saturated air-CO2 atmosphere (19:1) for 60 min with 100 µl/well serum-free medium containing 1 µM cyclosporin A, 1 µM probenicid, and 4 µM Fluo-3AM
previously dissolved in 20% pluronic acid (1 mg/ml stock). After
the loading procedure, the monolayers were washed four times at
room temperature with 150 µl/well 10 mM Hepes buffer, pH
7.4, containing 145 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM
MgCl2, 10 mM glucose, 1 µM cyclosporin A, and 1 µM probenicid (assay buffer), after which 180 µl of the same buffer was
added to each well, and the plates were used immediately. Chemokine-induced calcium mobilization and influx in DCCR2
receptor-expressing HEK 293 cells was monitored at room temperature by means of a fluorescent imaging plate reader (Molecular Devices Ltd., Menlo Park, CA). The excitation wavelength of
488 nm was supplied by a 3W argon laser (Coherent) set at 0.8-
1.2 W according to the level of cell confluency. Fluorescent
emission was measured at 512 nM. All assays were conducted at a
mean fluorescent baseline signal of 3.0-3.5 × 104 fluorescent cpm
per well and per plate. Lens aperture was maintained constant at
f/1.4, and shutter speed was set at 0.3-0.4 s. A total of 120 sampling points were collected for each well over 6-8-min periods.
Chemokine experiments were run by adding 20 µl of each chemokine at 10-fold the desired final concentration to each
well, and monitoring Fluo-3 fluorescence for a further 6 min. For
antagonist assays, 20 µl of each chemokine or pharmacological
agent of interest was added at 10-fold the desired final concentration to each well, followed by a 5-min incubation period after
which 20 µl of synthetic MIP-3 was added to each sample (final
[MIP-3
] = 100 nM), and fluorescence was monitored for an
additional 3 min. Sample injection speed was set at 175 µl/min,
and pipettor height at 180 µl. Under these conditions, maximal
signal amplitude was an additional 0.5-1.0 × 104 fluorescent
counts upon addition of 100 nM synthetic MIP-3
to wells containing only vehicle. Experiments with pertussis toxin-treated cells were conducted by preincubating DCCR2-expressing cells
with 500 nM pertussis toxin as previously described (35). For statistical analysis of fluorometry experiments, Student-Fisher unpaired bilateral t tests and analysis of variance using the Scheffe F
test criterion for unbalanced groups were used, where applicable.
The data represent the means ± SEM of at least four experiments
performed in single determinations.
Chemotaxis Assays.
Chemotaxis assays were performed in 48-well micro chemotaxis chambers (Neuroprobe, Inc., Cabin John,
MD). Neutrophils, monocytes, and DCs were suspended at 106
cells/ml, and T cells at 2 × 106 cells/ml, in RPMI medium containing 2 mM glutamine and 25 mM Hepes, and applied to the
top wells of the chamber. Standard 5-µm pore filters for monocytes, T cells, and DCs and 3-µm filters for neutrophils separated
the cells from the bottom chamber, which contained dilutions
of conditioned medium from cells transfected with either
pcDNA3.1(+) MIP-3-11, MIP-3
-16 or pcDNA3.1(+) CAT alone (mock transfected) or synthetic MIP-3
, at concentrations of 10
10-10
6 M. Chambers were incubated at 37°C for 20 min
(neutrophils), 60 min (monocytes and T cells), or 90 min (DCs),
after which time cells that had migrated onto the underside of the
filter were stained using Diff-Quik (Baxter Scientific, Inc., Unterschliesshe, Germany) and counted using an image analyzer or
five high power (×100) oil immersion fields. Human recombinant MCP-1, MCP-3, and IL-8 were used as positive controls in
the chemotaxis assays at a concentration of 100 nM. Each data
point was performed in triplicate and the results are representative
of at least three independent experiments using leukocytes from
different donors.
Radioligand Competition Binding Analysis. Binding assays were performed in triplicate on 105 HEK 293 cells transfected with DCCR2/pcDNA3.1(+) or with pcDNA3.1/CAT (as control), in Millipore 96-well filtration plates as described previously (26). Results are representative of at least three different experiments. The data obtained were curve-fitted to obtain the IC50 values using GraFit version 3.01 software (Erithicus Software Ltd., Staines, UK) according to a modified four-parameter logistic equation where F = B/Bmaxapp = 1 / (1 + [L]n / IC50) + background, where B = cpm bound, Bmaxapp = cpm bound in the absence of competing ligand, [L] is the concentration of competing ligand, and n is the slope of the curve.
A search of
the human EST databases revealed the presence of several
novel chemokine-like sequences (8). The full coding sequence of one of the EST sequences that appeared to encode a novel CC chemokine was cloned by reverse transcriptase PCR using primers based on the EMBL/GenBank/
DDBJ database accession number, D31065. Of the 16 cDNA libraries and 8 leukocyte cDNAs tested, D31065
cDNA was only detected in lung macrophages, lung DCs, and eosinophils. Sequencing of one of the resultant clones
(clone 11) from the macrophage PCR products revealed
that it was identical to the recently described MIP-3 and
LARC sequences (Fig. 1). In addition to the D31065 sequence, we also detected a population of PCR products
that contained an in-frame, three nucleotide deletion in the
putative signal sequence coding region. As the resultant amino acid deletion may effect the position of cleavage of
the signal peptide, we expressed both forms of MIP-3
,
MIP-3
clone 11 (wild type), and MIP-3
clone 16 (mutant) in HEK 293 cells. MIP-3
protein was purified from
conditioned medium 48 h after transfection. Amino acid
sequence analysis demonstrated that the NH2 terminus of
wild-type MIP-3
occurred at Ala 27. By contrast, the
NH2 terminus of the mutant MIP-3
was one amino
shorter starting at Ser 27.
Analysis of MIP-3
The expression of MIP-3 messenger RNA (mRNA) was analyzed in human multiple tissue Northern blots (Fig. 2 A). MIP-3
mRNA was highly
expressed in lung and in inflammed tissues such as tonsil
and appendix. Weaker expression was also detected, predominantly at mucosal sites and in lymphatic tissues: placenta, gall bladder, stomach, thymus, prostrate, testis, small
intestine, and colon. RT-PCR analysis of distinct leukocyte populations indicated MIP-3
mRNA present in lung
macrophages, DCs, and peripheral blood eosinophils (Fig.
2 B).
Chemoattractant Activity of MIP-3
Synthetic MIP-3 was
tested at concentrations of 10
6 to 10
10 M in chemotaxis
assays to characterize the leukocyte populations that responded to this chemokine (Fig. 3). MIP-3
was a potent chemoattractant for T lymphocytes and CD34+-derived
DCs, which migrated with typical bell-shaped dose-
response curves. The maximal chemotactic activity on T
cells was observed at 10
9 M, whereas on CD34+ DCs the
potency was 12.5-fold lower. No chemotactic activity was
observed with peripheral blood monocyte-derived DCs,
monocytes, or neutrophils. We also tested the chemotactic
activity of conditioned medium from HEK 293 cells transfected with wild-type MIP-3
(MIP-3
clone 11) or mutant MIP-3
(MIP-3
clone 16). The level of expression of both proteins (estimated from the HPLC peak height)
was similar and ~0.01-0.1 µg/ml. Whereas the media from
both transfectants was chemotactic when compared with
conditioned media from mock transfectants, the MIP-3
wild type appeared to be at least 2.5-fold more efficacious
than the mutant form.
Identification of Candidate MIP-3a Receptors Expressed on T Cells and DCs by Degenerate Oligonucleotide RT-PCR.
MIP-3 was
unable to displace the binding of specific chemokine
ligands to Chinese hamster ovary or HEK 293 cells stably
expressing the known chemokine receptors CCR1-5 and
CXCR1 and CXCR2 (data not shown). Therefore, we
used a degenerate oligonucleotide PCR strategy to identify
other candidate chemokine receptor-like cDNAs expressed
in these cells. Degenerate oligonucleotide primers based on
the conserved amino acid sequence found in the second intracellular loop and the seventh transmembrane domain of
the known human chemokine receptors were used in RT-PCR experiments on human lung DCs. PCR products
corresponding to the predicted size of ~500-550 bp were gel purified, subcloned, and sequenced. The majority of
the resultant clones corresponded to known chemokine receptors CCR1-5 and CXCR1-4. In addition, we also detected several cDNAs encoding orphan chemokine receptor-like sequences.
One of the orphan receptor sequences that we identified
in DCs, which we have called DCCR2, had earlier been
identified as GCY4, CKR-L3 (25), or STRL22 (24). RT-PCR on RNA extracted from distinct human leukocyte
populations indicated that DCCR2 mRNA expression was
restricted to T cells, CD34+ DCs, and lung DCs but not on
peripheral blood monocyte-derived DCs (Fig. 4). Full-length DCCR2 was subsequently cloned from human lung
DCs by RT-PCR using specific primers based on the full-length sequence given in the EMBL/GenBank/DDBJ database under accession number U48494. The resultant 1.1-kb
cDNA was subcloned into the mammalian cell expression
vector pcDNA3.1 and sequenced. One of the clones,
DCCR2-10, had an identical sequence to that deposited under U48494. The phylogenetic relationship of DCCR2
with other chemokine receptors is shown in Fig. 5. DCCR2
clusters with the CXC chemokine receptor family being
most closely related to another orphan receptor, EBI-1 (36)
(42% amino acid identity), which just before submission of
this manuscript, was reported to be CCR7 (37).
Functional Expression of DCCR2 in HEK 293 Cells.
It has
previously been shown that chemokine receptors typically
couple to phospholipase C (PLC) through a pertussis
toxin-sensitive G protein-dependent mechanism, which
subsequently leads to increased phosphatidyl inositol 4, 5, bisphosphate hydrolysis, and inositol 1, 4, 5 trisphosphate
(IP3)-dependent calcium mobilization (23). In view of this,
we attempted to identify the putative ligand(s) for DCCR2
by monitoring intracellular free calcium levels in monolayers of transiently transfected HEK 293 cells exposed to a
battery of 18 different human CC and CXC chemokines.
Of all the chemokines tested at 250 nM-1 µM concentrations (MCP-1, MCP-3, MIP-1, MIP-1
, MIP-3
, MIP-5,
RANTES, eotaxin, HCC-1, I-309, fractalkine, lymphotactin, SDF-1
, SDF-1
, IL-8, NAP-2, GRO
, and IP-10),
only MIP-3
induced a significant calcium response in these
preparations. As shown in Fig. 6, incubation of DCCR2
expressing HEK 293 cells with 100 nM MIP-3
promoted
a rapid, transient increase in cytosolic-free calcium, a response that was maximal within 25-30 s of application (from 2,230 ± 489 cpm to 7,541 ± 1,237 cpm at 25 s; n = 8, P <0.05 versus basal), and was sustained for up to 5 min
thereafter (4,234 ± 869 cpm at 3.5 min, n = 8). Furthermore, the response was dose dependent, exhibiting a half-maximal effective concentration (EC50) of 100-200 nM.
In view of the observations that many CC chemokines
bind to more than one chemokine receptor in vitro, but
that only MIP-3 promotes significant increases in cytosolic-free calcium in our preparations, we further attempted
to detect chemokine-DCCR2 interactions by submitting
transfected HEK 293 cells to desensitization protocols in
which the cells were exposed for 5 min to 500 nM to 1 µM
concentrations of 14 CC and CXC chemokines before being challenged with 100 nM MIP-3
. As shown in Fig. 7
A, preexposure of cells to 1 µM concentrations of IL-8,
GRO
, NAP-2, or IP-10 had no significant effect on the calcium signal induced by 100 nM MIP-3
(n = 4). Preincubation with 500 nM to 1 µM concentrations of nine human CC chemokines (MCP-1, MIP-1
, MIP-1
, MIP-5,
RANTES, eotaxin, HCC-1, I-309, and fractalkine), also
had no effect significant on MIP-3
-induced calcium responses (n = 4; see Fig. 7 B), although preincubation with
both 1 µM RANTES and 1 µM HCC-1 did decrease the
MIP-3
-induced calcium response in two out of four experiments. Indeed, only preincubation with 1 µM MIP-3
led to a significant decrease in MIP-3
-induced calcium
responses. The effect appeared to correspond to near maximal receptor desensitization (97 ± 12% inhibition of calcium response; n = 4; see Fig. 7 B), and was dose dependent, with MIP-3
exhibiting a half-maximal inhibiting
concentration (IC50) of 1-10 nM (n = 4; see Fig. 7 C).
We also investigated the cell signaling pathways underlying MIP-3-induced calcium responses in DCCR2-
expressing HEK 293 cells by monitoring the effect of MIP-3
on cytosolic free calcium in pertussis toxin-treated
monolayers, a procedure that leads to the inhibition of G
protein effector coupling by inactivational ADP-ribosylation. As shown in Fig. 8 A, a 24-h pretreatment of DCCR2 receptor-expressing cells with 500 nM pertussis
toxin led to a marked decrease in the calcium responses induced by either 100 or 500 nM MIP-3
(74% and 61% inhibition, respectively; n = 4, P <0.05), a result suggesting
that the G protein-dependent activation of a PLC is implicated in MIP-3
-induced calcium responses in this system.
Consistent with this last hypothesis, MIP-3
-induced calcium responses were only partially inhibited when studied
in the presence of 1.6 mM extracellular EGTA (30% inhibition; n = 4, P <0.05; see Fig. 8 B), a result suggesting
that a substantial portion of the MIP-3
-induced calcium
response is derived from the IP3-mediated mobilization of
calcium from intracellular stores. In agreement with this
possibility, MIP-3
-induced calcium responses were reduced in the presence of 10 µM of the selective PLC inhibitor, U-73122 (38), as well as in cells pretreated with 1 µM
4b PMA, a widely used experimental condition known to
activate protein kinase C, and consequently inhibit PLC
through a negative feedback process (39). Consistent with
these last results, preincubation of DCCR2 receptor-
expressing cells with the Ca2+-ATPase inhibitors thapsigargin and cyclopiazonate (40, 41) promoted a significant inhibition of the calcium response induced by MIP-3
(55 and 60% inhibition at 1 and 120 µM, respectively; n = 4, P <0.05; see Fig. 8 D).
Binding Studies.
We also tested the ability of the
DCCR2-transfected HEK 293 cells to bind to radiolabeled
synthetic MIP-3. In competition binding assays, synthetic
MIP-3
was able to displace [I125]-MIP-3
with an IC50
value of ~12 nM (Fig. 9).
Chemokines play an important role in the trafficking of
immune cells around the body and in diverse physiological
processes such as inflammation, infection, hematopoeisis,
and development (42, 43). Their actions are mediated
through a family of highly homologous, G protein-coupled, seven-transmembrane receptors. Here, we report the
cloning and functional characterization of a novel chemokine receptor from lung DCs that is highly specific for the
recently described CC chemokine, MIP-3.
MIP-3 was first identified in EST databases (8, 21, 22).
We have shown by Northern blot analysis that the mRNA
for this chemokine is highly expressed in potentially inflamed tissues such as tonsil and appendix, and in lymphoid
and mucosal tissues, particularly in lung. Despite the earlier
identification of LARC as a liver chemokine (22), we
found only weak, barely detectable expression in liver on
two independent Northern blots, suggesting that the expression may vary among individuals, possibly depending on immune status. The specific cell types that express MIP-3
appear to be macrophages, eosinophils, and DCs, as
shown by RT-PCR experiments. These cell populations
are known to be well represented at mucosal sites. In lung,
macrophages are found in large numbers both within the
airway lumen and the lung interstitium. They are the first
line of defense against invading organisms and allergens and
play an important role in innate and acquired immunity
(44, 45). Lung DCs reside within the airway epithelium
and are the most potent APCs known (46, 47). These cells
uptake, process, and present foreign antigen or allergens to
T cells either in situ or at local lymph nodes, resulting in T
cell activation and proliferation. Eosinophils are not usually
found in large numbers in normal human lung. However,
they do occur at other mucosal sites, notably in the gastro-
intestinal tract of normal individuals, where they are thought
to play a major role in host defense against mucosal pathogens (48). Thus, the finding that MIP-3
mRNA is constitutively expressed at high levels in these cell types suggest that MIP-3
may play an important role in the rapid recruitment of inflammatory cells to potential sites of infection.
To identify the specific cell populations which responded to MIP-3, and for characterization of the MIP-3
receptor, we used chemically synthesized protein. Synthetic MIP-3
was chemotactic for T cells but not for
monocytes and neutrophils. The maximal chemotactic effect was observed at around 10
9 M. MIP-3
was also
chemotactic for CD34+-derived DCs but was an order of
magnitude less potent than on T cells. Peripheral blood
monocyte-derived DCs did not migrate in response to
MIP-3
. Differences in chemokine responsiveness of CD34+
derived DCs and peripheral blood monocyte-derived DCs
have been reported in the literature (31, 49). These differences in the chemotactic activity may be a consequence of
different degrees of maturation of the DCs or reflect important functional differences between DCs obtained from
distinct sources. We have preliminary data to show that
MIP-3
is chemotactic on lung DCs between 10
7 and
10
9 M but due to the difficulties in obtaining sufficient
numbers of lung DCs to do statistically significant numbers
of chemotaxis assays, we are unable to present this data at
present.
We and others have shown that MIP-3 does not act
through any of the known CC chemokine receptors or
through CXCR1 or CXCR2 (CXCR3, CXCR4, and
DARC were not tested). We have cloned a number of orphan chemokine receptor-like molecules from lung DCs in
addition to the published chemokine receptors. One of these
orphans, DCCR2, variously known as GCY-4, STRL-22,
or CKRL-3 was shown by RT-PCR to be expressed in T
cells and certain DC populations, an mRNA expression
pattern consistent the MIP-3
responsiveness in chemotaxis assays.
We also demonstrated that synthetic MIP-3 is able to
mobilize intracellular Ca2+ in HEK 293 cells transiently expressing DCCR2. The maximal effect of MIP-3
was observed at 1 µM and could not be agonized or antagonized by at least 18 other human chemokines tested, thus confirming the specificity of this receptor. In common with
other CC chemokine receptors, DCCR2-dependent calcium responses appear to result from both EDTA-sensitive
calcium influx on the one hand, and pertussis toxin-sensitive
PLC activation on the other. Furthermore, the results obtained in the presence of U-73122 and in PMA-treated cells suggest that IP3-mediated calcium mobilization is at
the basis of this second element of the response, particularly
as depletion of intracellular calcium stores with either
thapsigargin or cyclopiazonate also inhibit the MIP-3
-
induced increases in cytosolic free calcium. Interestingly,
the EC50 of the Ca2+ response was much higher than the
concentration of MIP-3
required to produce a maximal
chemotactic effect in T cells and CD34+ derived DCs, and
the IC50 of radiolabeled MIP-3
binding to HEK 293 cells
transiently expressing DCCR2. This may be due to the fact
that different concentrations of chemokine may be required for different physiological functions as has been observed for other chemokines (50). Earlier studies have
shown that modifications at the NH2 terminus of some
chemokines can alter their activity; for example, the addition of methionine to the NH2 terminus of RANTES
changes it from an agonist of CC chemokine receptors, to an antagonist, without changing its receptor binding properties (51). While it is clear that the NH2 terminus of the
synthetic MIP-3
used in this study contains an additional
three amino acids compared with the mature protein expressed in HEK 293 cells, our results would indicate that
this modification is only likely to change the binding affinity and potency in different functional assays rather than receptor specificity. In support of this, recent studies on mature LARC produced in insect cells indicated that it failed
to bind to any of the known chemokine receptors (22) suggesting that the synthetic MIP-3
used here behaves in a
manner analogous to the recombinantly expressed protein.
However, it remains to be seen whether there are other as
yet uncharacterized orphan receptors that bind MIP-3
or,
indeed, whether other ligands for DCCR2 will be found
amongst the ever-increasing number of chemokines.
DCCR2 shows several features that make it distinct from
other CC chemokine receptors: the gene for DCCR2 resides
on chromosome 6q27 (24); all of the other CC chemokine
receptors identified to date have been localized on chromosome 3p21-24 (52); so far, it appears to be highly selective for a single ligand (MIP-3), unlike the other CC
chemokine receptors, which are activated by at least two to
three chemokine ligands; and the constitutive mRNA expression of DCCR2 seems to be highly restricted to T cells and DCs. The finding of both ligand and receptor in DCs
is perhaps also indicative of an autocrine loop existing in
these cells, suggesting that the ligand, MIP-3
, may have
functions in addition to being a chemoattractant.
The primary role of DCs is to take up and process foreign antigens, which are then presented to naive T cells, thus
initiating the immune cascade. During inflammation, the
numbers of DCs resident in tissues increases and there is
also increased trafficking of DCs from the tissue to the local
lymph nodes (53). This ensures rapid resolution of the infection or inflammation. However, excessive local T cell
activation by DCs may be an important contributory factor
in allergic diseases such as asthma. As DCCR2 shows relatively restricted expression to T cells and DCs, and is
highly specific for MIP-3, this ligand-receptor pair is
likely to be an important therapeutic target for modulation
of the immune response.
Address correspondence to Dr. C.A. Power, Geneva Biomedical Research Institute, GlaxoWellcome Research and Development S.A., 14, chemin des Aulx, 1228, Plan-les-Ouates, Geneva, Switzerland. Phone: 00-41-22-7069-752; FAX: 00-41-22-7946-965; E-mail: CAP15123{at}ggr.co.uk
Received for publication 5 June 1997 and in revised form 3 July 1997.
1 Abbreviations used in this paper: CAT, cloramphenicol acetyl transferase; DARC, Duffy antigen receptor; DCs, dendritic cells; DCCR2, dendritic cell chemokine receptor 2; EST, expressed sequence tag; HEK, human embryonic kidney; LAMCs, loosely adherent mononuclear cells; LARC, liver and activation-regulated chemokine; mRNA, messenger RNA; PLC, phospholipase C; RT, reverse transcriptase; SDF-1, stromal cell- derived factor-1.The authors thank Dr. J.-P. Aubry for FACS®; F. Borlat, L. Menoud, and Y. Cambet for technical assistance; D. Bertschy and M. Huguenin for DNA sequencing; E. Magenat for protein sequencing; and C. Hebert for photography.
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