From the
Eosinophils undergo chemotaxis, degranulate, and exhibit
[Ca
The types of white blood cells that appear in inflammatory
infiltrates can differ markedly depending in part on the identity of
the inflammatory irritant and the duration of irritation. The host
factors responsible for these differences are likely to be cell
type-specific or selective chemoattractants and chemoattractant
receptors. CC and CXC chemokines are two related families of relatively
selective leukocyte chemoattractants. CC chemokines are all potent
monocyte chemoattractants in vitro but differ in their
capacity to activate neutrophils, lymphocytes, basophils, and
eosinophils (reviewed in Ref. 1). In particular, human
MIP-1
The receptors responsible for CC
chemokine action on eosinophils have not been defined. By inspecting
cross-desensitization patterns for chemokine-induced calcium
transients, Dahinden et al. (4) have proposed that eosinophils
have specific receptors for RANTES and MCP-3 and a shared receptor for
MIP-1
cDNAs for three human
leukocyte CC chemokine receptors have been cloned, CC CKR1, CC CKR2A,
and CC CKR2B (also known as MCP-1 receptors A and B), but their
properties are not fully consistent with eosinophil responses to CC
chemokines(8, 9, 10, 11) . MIP-1
The CC CKR3 cDNA is 1.6 kb in length. The ORF encodes a predicted
protein of 355 amino acids that is identical in length and 63%
identical in sequence with CC CKR1. CC CKR3 has 51% identity with CC
CKR2B (Fig. 1) but only 31% identity with the CXC chemokine
receptors and IL-8 receptors A and
B(8, 9, 10, 11, 16, 17) .
The amino acid positions that differ between CC CKR1 and CC CKR3 are
found mostly in the putative extracellular domains and adjacent
portions of the transmembrane domains (Fig. 1); the predicted
intracellular C-terminal segments are also divergent but have a high
content of serine residues that may be sites for receptor
phosphorylation as they are in rhodopsin and the
The factors that account for these
differences are not clear but could include distinct genes for novel CC
chemokine receptors. Alternatively, cell type-specific accessory
factors could exist that modulate agonist and desensitization
properties for the receptors that are already known. It is known, for
example, that GRO
Since MIP-1
While the distinctive RNA distribution
patterns of the cloned CC chemokine receptors are a useful starting
point, additional functional studies will be required to fully
delineate the importance of these receptors for chemotaxis,
degranulation, and other responses by specific types of leukocytes
during inflammatory responses in vivo. Nevertheless, based on
the data reported here, it is reasonable to hypothesize that CC CKR3
may be an important host factor regulating eosinophil accumulation at
body sites undergoing allergic reactions, metazoan infestation, or
other types of inflammation where eosinophils are present in large
numbers.
We thank S. Mawhorter for providing purified
eosinophils and H. Lee Tiffany for excellent technical assistance.
Note Added in Proof-The gene symbol for the CC CKR3 gene is CMKBR3 (P. McAlpine, personal communication). A mouse gene
designated scya3r-rs2 has been identified that appears to be
the orthologue to CMKBR3(26) .
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
]
changes in
response to the human CC chemokines macrophage inflammatory protein
(MIP)-1
, regulated on activation, normal T expressed and secreted
(RANTES), and monocyte chemoattractant protein-3 (MCP-3), but the
receptors involved have not been defined. We have isolated a human cDNA
encoding the first eosinophil-selective chemokine receptor, designated
CC chemokine receptor 3 (CC CKR3). CC CKR3 is a seven-transmembrane
domain G protein-coupled receptor most closely related to the
previously reported monocyte- and neutrophil-selective receptor CC CKR1
(also known as the MIP-1
/RANTES receptor). When
[Ca
]
changes were
monitored in stably transfected human embryonic kidney 293 cells,
MIP-1
and RANTES were both potent agonists for CC CKR3 and CC
CKR1. However, MIP-1
was also an agonist for CC CKR3 but not CC
CKR1; MCP-3 was an agonist for CC CKR1 but not CC CKR3. CC CKR3 may be
one of the host factors responsible for selective recruitment of
eosinophils to sites of inflammation.
,
(
)RANTES, and MCP-3 and guinea pig
eotaxin are potent eosinophil chemoattractants, whereas other known CC
chemokines are not(2, 3, 4, 5) . The
best characterized CXC chemokine, interleukin-8 (IL-8), is a strong
neutrophil chemoattractant but has only modest activity for
eosinophils(1, 6) .
, RANTES, and MCP-3. Van Riper et al.(7) have identified a shared functional binding site for
MIP-1
and RANTES on HL-60 cell-derived eosinophil-like cells, but
MCP-3 binding to this site has not yet been tested. Eotaxin competes
for
I-human RANTES binding to guinea pig eosinophils,
suggesting a shared receptor(5) .
and RANTES are effective agonists for CC CKR1; however, its RNA is
scarce in eosinophils(8, 9, 12) . Much higher
expression is found in neutrophils, monocytes, and
lymphocytes(10, 12) . MCP-1 is an agonist for CC CKR2A
and -2B, but it does not activate eosinophils(4, 11) .
Moreover, CC CKR2 RNA is expressed in monocytes but not in
eosinophils(12) . Together with the CXC chemokine receptors, the
CC chemokine receptors form a subgroup of the rhodopsin superfamily of
seven-transmembrane domain receptors(13) . Here we characterize
the first eosinophil-selective member of this family.
Gene and cDNA Cloning
Construction of a
gt11 cDNA library prepared from lipopolysaccharide-stimulated
peripheral blood monocytes has been previously described(14) .
The library was screened by plaque hybridization with the full-length
open reading frame (ORF) of CC CKR2B produced by polymerase chain
reaction and labeled with [
-
P]dCTP using a
random primer labeling kit (Boehringer Mannheim). Plaque lifts were
incubated in 10
cpm/probe/ml of hybridization buffer for 12
h and then were washed at 55 °C in 5
SSPE for 15 min. cDNA
inserts of purified phage DNA were excised by SalI and SfiI double digestion, blunt ended with Pfu DNA
polymerase, subcloned into the EcoRV site of pBluescript II SK
(Stratagene, La Jolla, CA), and sequenced on both strands. cDNAs were
then subcloned between the NheI and XhoI sites of the
mammalian expression vector pREP9 (Invitrogen, San Diego, CA). Human
embryonic kidney (HEK) 293 cells (10
) grown to log phase in
Dulbecco's modified Eagle's medium and 10% fetal bovine
serum were electroporated with 20 µg of plasmid DNA, and
G418-resistant colonies were picked and expanded. The p4 cDNA (8) encoding CC CKR1 was subcloned into the NotI and XhoI sites of pCEP4 (Invitrogen), and transfected HEK 293
cells were selected in 250 µg/ml hygromycin.
DNA Analysis
Human genomic DNA was analyzed by
restriction enzyme cleavage and Southern hybridization as described
previously(8) .
RNA Analysis
Total RNA was prepared using an RNA
isolation kit (Stratagene, La Jolla, CA) from human peripheral blood
neutrophils isolated by Hypaque-Ficoll differential centrifugation
(>95% neutrophils), from human blood monocytes separated from
lymphocytes by adherence to plastic for 18 h, and from human blood
eosinophils isolated from a healthy individual with a >10-year
history of stable hypereosinophilia (>99% eosinophils) as described
previously (15). RNA samples were analyzed by Northern blot
hybridization exactly as described previously(15) .
Ligand Binding Analysis
10 transfected
cells were incubated in duplicate with 0.1-0.5 nM
I-labeled RANTES, MCP-1, MIP-1
, or MIP-1
(specific activity,
2200 Ci/mmol, DuPont NEN) and varying
concentrations of unlabeled recombinant human chemokines (Peprotech,
Rocky Hill, NJ) in 200 µl of binding medium (RPMI 1640 with 1 mg/ml
bovine serum albumin and 25 mM HEPES, pH 7.4). After
incubation for 2 h at 4 °C, cells were pelleted through a 10%
sucrose/phosphate-buffered saline cushion, and specific binding was
determined by the difference in counts in the presence and absence of a
500-fold molar excess of unlabeled chemokine.
Intracellular [Ca
Cells (10] Measurements
/ml) were incubated in
Hanks' buffered saline solution with Ca
and
Mg
supplemented with 10 mM HEPES, pH 7.4
(HBSS) containing 2.5 µM Fura-2/AM (Molecular Probes,
Eugene, OR) for 60 min at 37 °C in the dark. Cells were washed
twice with HBSS and resuspended at 2
10
cells/ml.
Two ml were placed in a continuously stirred cuvette at 37 °C in a
fluorimeter (Photon Technology Inc., South Brunswick, NJ). Fluorescence
was monitored at
= 340 nm,
= 380 nm, and
= 510 nm. The data
were recorded as the relative ratio of fluorescence excited at 340 and
380 nm. Data were collected every 200 ms.
Sequence Analysis of CC CKR3
Out of 6 positive
human monocyte cDNA clones that cross-hybridized with a CC CKR2B ORF
probe at low stringency, 2 were for CC CKR1, 1 was for CC CKR2B, 2 were
for novel putative chemokine receptors, and 1 was for a cDNA unrelated
to chemokine receptors. Here we detail the properties of one of the two
novel putative chemokine receptors, which we have designated CC CKR3.
-adrenergic receptor(18) . Like CC CKR1 and all
other known chemokine receptors, the CC CKR3 sequence is acidic in the N-terminal segment before the first putative transmembrane
domain, containing a net charge of -4. The second extracellular
loop is also highly acidic (net charge of -5), whereas for CC
CKR1 the corresponding region has a net charge of +3. Like all
other known chemokine receptors, CC CKR3 has conserved cysteine
residues in the N-terminal segment and the third predicted
extracellular loop that could form a disulfide bond (13). These
cysteines are less frequently found in other G protein-coupled
receptors(19) . Unlike other chemokine receptors, CC CKR3 lacks
a consensus sequence for N-linked glycosylation. The proline
in a proline-cysteine motif conserved in the N-terminal
segment of other chemokine receptors is leucine in CC CKR3.
Figure 1:
Alignment of amino acid sequences
deduced from cDNAs for CC CKR1, CC CKR2B, and CC CKR3. Verticalbars indicate identical residues for each adjacent
sequence position. Arabicnumbers enumerate the CC
CKR3 sequence and are left-justified. Dashes indicate gaps
that were inserted to optimize the alignment. The locations of
predicted membrane-spanning segments I-VII are noted. Openboxes designate predicted sites for N-linked glycosylation. The DNA and proteins sequences have
been deposited in GenBank and have been given the accession number
U28694.
Hybridization of the CC CKR3 cDNA to total human genomic DNA
digested with PstI, EcoRI, and HindIII
revealed one strongly hybridizing band in each case (Fig. 2A), suggesting that it is the product of a small,
single-copy gene. The 1.8-kb HindIII band was also seen in
genomic DNA hybridized under low stringency conditions with a CC CKR1
cDNA probe due to cross-hybridization to CC CKR3 (9).(
)
Figure 2:
Analysis of the CC CKR3 gene and RNA. A, gene. 5 µg of total human genomic DNA was digested with
the restriction enzymes indicated at the top of each lane, gel-fractionated, and blot-hybridized with the CC CKR3
cDNA. The blot was washed at 65 °C in 0.2 SSPE for 1 h and
then exposed to XAR-2 film with an intensifying screen at -80
°C for 5 days. Size markers in kb are indicated at the left. B, distribution of mRNA encoding CC CKR3 in
human leukocytes. A Nytran blot containing 10 µg of total RNA from
peripheral blood-derived human neutrophils (N), monocytes (M), and eosinophils (E) was hybridized with a CC
CKR3 cDNA probe. The blot was washed at 65 °C in 0.2
SSPE
for 1 h and then exposed to XAR-2 film with an intensifying screen at
-80 °C for the times indicated at the bottom of each panel. The position of ribosomal bands is indicated at the left.
Distribution of CC CKR3 RNA
Two size
classes of CC CKR3 mRNA were detected in human peripheral blood-derived
eosinophils (Fig. 2B). The major species is 1.6 kb,
similar in length to the CC CKR3 cDNA that we cloned. A minor 4-kb
species is also present. Faint 1.6-kb bands were visible in neutrophil
and monocyte samples only after prolonged exposures. When the same blot
was probed with CC CKR1 cDNA, a reciprocal banding pattern was
detected: large amounts of a 3-kb transcript in neutrophil and monocyte
lanes and trace amounts in the eosinophil lane(12) . CC CKR1
mRNA has also been detected in peripheral blood-derived lymphocytes and Staphylococcus aureus Cowan strain-activated tonsillar B cells (8, 10) and in small amounts in heart, kidney, skeletal
muscle, pancreas, and brain but in larger amounts in placenta, lung,
and liver(12) . We have not been able to detect CC CKR3
transcripts in these solid organs (not shown). When the same phagocyte
blot was probed with the CC CKR2B ORF, a 3.5-kb transcript was seen
only in the monocyte lane(12) . Thus, CC CKR1, -2, and -3 are
differentially expressed in a cell type-specific pattern in human
peripheral blood leukocytes.
Agonists for CC CKR3
Since MIP-1, RANTES, and
MCP-3 are the only known human CC chemokines that activate eosinophils,
they were the best candidate agonists for CC
CKR3(2, 3, 4, 7) . Chemotactic responses
to chemokines are associated with rapid and transient elevations of
[Ca
]
, which can be
used as a convenient indicator of receptor usage. Neither untransfected
nor mock-transfected and selected HEK 293 cells responded to any of the
chemokines tested. In contrast, when three independent HEK 293 cell
clones stably transfected with CC CKR3 cDNA were tested, all three
exhibited [Ca
]
transients in response to MIP-1
, RANTES, and MIP-1
but not in response to MCP-1, MCP-2, MCP-3, IL-8, or
IP-10, all
tested at 100 nM (Fig. 3A). The rank order of
potency was MIP-1
> RANTES > MIP-1
(Fig. 3B). CC CKR3 is the first known MIP-1
receptor to be cloned. As previously reported, HEK 293 cells stably
transfected with CC CKR1 also responded to MIP-1
and
RANTES(9, 20) . However, unlike CC CKR3, CC
CKR1-transfected cells also responded to MCP-3 but not to MIP-1
at
100 nM (Fig. 3A). CC CKR1 is the first known
MCP-3 receptor to be cloned. The relationship of CC CKR1 to CC CKR3 is
analogous to the relationship of IL-8 receptor A to IL-8 receptor B, in
that both are pairs of receptors with highly related sequences and an
overlapping but non-identical set of agonists restricted to either CC
or CXC chemokines(16, 17) . However, IL-8 receptors A
and B are both expressed selectively at high levels in the same cell
type, the neutrophil(15) .
Figure 3:
Agonists for CC CKR3. A,
selectivity for CC CKR3 versus CC CKR1.
[Ca] was monitored by ratio fluorescence of
Fura-2-loaded HEK 293 cells stably transfected with plasmids containing
the cDNAs indicated at the top of each column. Cells
were stimulated at the time indicated by the arrows with the
chemokine indicated at the left of each row of tracings, at 100 nM. The tracings shown are from one
experiment representative of at least three experiments with three
separate clones for CC CKR3 and one clone for CC CKR1. B, rank
order of potency of agonists for CC CKR3. The magnitude of the peak of
the calcium transient elicited by the indicated concentration of human
chemokines from HEK 293 cells stably expressing the CC CKR3 is shown.
Each data point represents the peak of one tracing. The data are from a
single experiment representative of at least three separate
experiments.
Desensitization of CC CKR3
After activation,
chemokine receptors have altered sensitivity to repeated stimulation
with the activating agonist and other
agonists(1, 4, 9, 20) . When CC CKR3
transfectants were sequentially stimulated with 100 nM of
various combinations of MIP-1, MIP-1
, RANTES, and MCP-1, the
following results were obtained (Fig. 4). MCP-1 had no effect on
the responses to MIP-1
, MIP-1
, or RANTES. MIP-1
,
MIP-1
, and RANTES completely desensitized the homologous response.
MIP-1
and RANTES completely cross-desensitized the response to
MIP-1
, but MIP-1
only partially cross-desensitized the
response to MIP-1
and RANTES. Finally, RANTES and MIP-1
partially cross-desensitized the responses to each other. This provides
additional evidence for a functional interaction of MIP-1
,
MIP-1
, and RANTES with the same receptor, CC CKR3.
Figure 4:
Desensitization of calcium transients in
HEK 293 cells stably transfected with CC CKR3. Ratio fluorescence was
monitored from Fura-2-loaded cells before and during sequential
addition of chemokines at 100 nM at the times indicated by the arrows. The first stimulus added (S1) for each
tracing is indicated at the left of the row in which
it is found. The second stimulus added (S2) is indicated at
the top of the column in which the tracing is found.
The tracings are from a single experiment representative of three
separate experiments.
It is
important to note that the [Ca]
changes in response to CC chemokines in eosinophils differ
significantly from those obtained with CC CKR1 and CC CKR3 in
transfected HEK 293 cells (4, 9) (Fig. 4). MIP-1
more
effectively desensitizes the CC CKR1 and CC CKR3 RANTES responses than
the eosinophil RANTES response. Conversely, RANTES more effectively
desensitizes the eosinophil MIP-1
response than the CC CKR1 and CC
CKR3 MIP-1
responses. Finally, MIP-1
is a potent agonist for
CC CKR3 but not for eosinophils. Neutrophil and monocyte responses to
MIP-1
and RANTES also differ from those found for the cloned
receptors. For neutrophils, responses to MIP-1
and RANTES can be
completely cross-desensitized, yet the magnitude of the neutrophil
RANTES response is half that of the neutrophil MIP-1
response at
saturating concentrations, whereas for CC CKR1 and CC CKR3
transfectants they are similar(8, 9)
(Fig. 3). RANTES attenuates both the CC CKR1 and CC CKR3
MIP-1
response in transfected cells but not the monocyte
MIP-1
response (9, 21).
does not effectively cross-desensitize
[Ca
]
changes in
response to IL-8 in HEK 293 cells transfected with IL-8 receptor B,
whereas IL-8 completely cross-desensitizes neutrophil responses to
GRO
, which are thought to be mediated by IL-8 receptor
B(22, 23) .
, RANTES, and MIP-1
are agonists for CC CKR3, they must bind to it. Nevertheless we have
not yet been able to demonstrate specific binding of
I-MIP-1
and -RANTES to CC CKR3-transfected HEK 293
cells using as much as 0.5 nM radioligand on 2 million
transfected cells. We have used the same conditions to demonstrate
specific binding of
I-MIP-1
to CC CKR1-transfected
HEK 293 cells (Fig. 5). This suggests that MIP-1
,
MIP-1
, and RANTES activate CC CKR3 via low affinity binding
interactions. It is possible that CC CKR3 is more selective for
another, as yet untested, CC chemokine such as eotaxin(5) .
Human eotaxin has not been identified yet. The high affinity binding of
I-MIP-1
and -RANTES to a shared site reported for
HL-60-derived eosinophils is most likely due to a receptor different
from CC CKR3, perhaps CC CKR1.
Figure 5:
Specific binding of chemokines to HEK 293
cells stably transfected with human CC chemokine receptor cDNAs. Cells
stably transfected with the indicated human cDNAs were incubated in
duplicate with 0.1 nMI-human MIP-1
in the
presence or absence of 50 nM human MIP-1
at 4 °C. The
total counts bound for CCCKR3 were not significantly different from
untransfected cells (not shown). Data are representative of two
separate experiments with three separate CC CKR3
clones.
The present and previous studies
strongly suggest that normal human monocytes and eosinophils respond to
MIP-1 and RANTES via two MIP-1
/RANTES receptors, CC CKR1 and
CC CKR3. The relative RNA distributions suggest that CC CKR1 functions
principally, but not exclusively, in monocytes, and CC CKR3 functions
principally, but not exclusively, in eosinophils. RNA for both CC CKR1
and CC CKR3 is expressed in neutrophils, but the functional importance
is unclear since these cells do not chemotax or degranulate to
MIP-1
and RANTES (24). CC CKR1 may also be responsible in part for
the responses of monocytes and eosinophils to
MCP-3(4, 25) . Since RNA for CC CKR1 is present in
neutrophils, we predict that MCP-3 can elicit
[Ca
]
changes in
neutrophils, although this has not been tested yet. The functional
significance for eosinophils of the response of CC CKR3 to MIP-1
is unclear at present.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.