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The Human Specific CCR1 Antagonist CP-481,715 Inhibits
Cell Infiltration and Inflammatory Responses in Human CCR1
Transgenic Mice
Ronald P. Gladue,
1
Susan H. Cole, Marsha L. Roach, Laurie A. Tylaska, Robin T. Nelson,
Richard M. Shepard, John D. McNeish, Kevin T. Ogborne, and Kuldeep S. Neote
We previously described the in vitro characteristics of the potent and selective CCR1 antagonist, CP-481,715. In addition to being
selective for CCR1 vs other chemokine receptors, CP-481,715 is also specific for human CCR1 (hCCR1), preventing its evaluation
in classical animal models. To address this, we generated mice whereby murine CCR1 was replaced by hCCR1 (knockin) and used
these animals to assess the anti-inflammatory properties of CP-481,715. Cells isolated from hCCR1 knockin mice were shown to
express hCCR1 and migrate in response to both murine CCR1 and hCCR1 ligands. Furthermore, this migration is inhibited by
CP-481,715 at dose levels comparable to those obtained with human cells. In animal models of cell infiltration, CP-481,715
inhibited CCL3-induced neutrophil infiltration into skin or into an air pouch with an ED
50
of 0.2 mg/kg. CP-481,715 did not inhibit
cell infiltration in wild-type animals expressing murine CCR1. In a more generalized model of inflammation, delayed-type hy-
persensitivity, CP-481,715 significantly inhibited footpad swelling and decreased the amount of IFN-
␥
and IL-2 produced by
isolated spleen cells from sensitized animals. It did not, however, induce tolerance to a subsequent challenge. These studies
illustrate the utility of hCCR1 knockin animals to assess the activity of human specific CCR1 antagonists; demonstrate the ability
of the CCR1 antagonist CP-481,715 to inhibit cell infiltration, inflammation, and Th1 cytokine responses in these animals; and
suggest that CP-481,715 may be useful to modulate inflammatory responses in human disease. The Journal of Immunology, 2006,
176: 3141–3148.
Leukocyte infiltration into inflammatory sites is believed to
be regulated by 8- to 10-kDa proteins known as chemo-
kines. These chemokines are classified into four groups,
depending on the spacing between two N-terminal cysteine resi-
dues, and are designated CC, CXC, XC, and CX
3
C chemokines.
The therapeutic potential of inhibiting chemokines or their recep-
tors is supported by their enhanced expression in human disease,
numerous studies in animal models, and, in some instances, ge-
netic association studies (1– 4). These reports have prompted the
identification and characterization of chemokine receptor antago-
nists, several of which are currently undergoing clinical trials (5).
One chemokine receptor thought to play a crucial role in several
diseases is CCR1. CCR1 is expressed on monocytes, T cells, den-
dritic cells, and, in some cases, neutrophils (6 –9), and interacts
with at least eight different ligands, including CCL3 (MIP-1
␣
),
CCL5 (RANTES), CCL7 (MCP-3), CCL14 (hemofiltrate C-C che-
mokine-1), CCL8 (MCP-2), CCL15 (leukotactin-1), CCL23 (my-
eloid progenitor inhibitory factor-1), and hemofiltrate C-C chemo-
kine-4 (CCL16) (10 –12). These ligands have been shown to have
potent chemotactic activity in vitro (9), and in some cases in vivo
in which intradermal injection of CCL3 or CCL5 into human sub-
jects induced a robust cell infiltration (8, 13). In addition to me-
diating cell migration, CCR1 signaling has been shown to up-reg-
ulate integrins such as Mac-1 (CD11b), thus causing the firm
adherence of leukocytes to the endothelium (14). CCR1 signaling
may also contribute to tissue damage and inflammation through the
enhancement of T cell activation (15), regulation of Th1/Th2 cy-
tokine polarization (16, 17), and stimulation of macrophage func-
tion (18) and protease secretion (14, 19, 20). Taken together, these
properties support CCR1 as an attractive therapeutic target to mod-
ulate leukocyte infiltration and decrease the associated tissue dam-
age common to many autoimmune diseases.
Numerous animal disease models have shown that inhibition of
CCR1 or its ligands abrogates disease. These data prompted dis-
covery efforts to identify small molecular weight mass CCR1 an-
tagonists and led to the identification of CP-481,715 (14).
CP-481,715 is a potent CCR1 antagonist that retains activity in
human whole blood. In addition to being selective for CCR1 as
compared with other G protein-coupled receptors, CP-481,715 is
also selective for the human CCR1 (hCCR1)
2
receptor, preventing
its assessment in classical animal models (14). To overcome this
obstacle, we generated mice that had murine CCR1 replaced by
hCCR1 and demonstrate the ability of CP-481,715 to inhibit in
vivo inflammatory responses in these animals at clinically achiev-
able dose levels.
Materials and Methods
Materials
The CCR1 antagonist, CP-481,715 (quinoxaline-2-carboxylic acid [4(R)-
carbamoyl-1(S)-(3-fluorobenzyl)-2(S),7-dihydroxy-7-methyl-octyl]amide),
Pfizer Global Research and Development, Department of Immunology, Groton, CT
06340
Received for publication September 13, 2005. Accepted for publication November
30, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Address correspondence and reprint requests to Dr. Ronald P. Gladue, Associate
Research Fellow, Pfizer Global Research and Development, Department of Immu-
nology, MS 8220-2410, Eastern Point Road, Groton, CT 06340. E-mail address:
Ronald.P.Gladue@Pfizer.com
2
Abbreviations used in this paper: hCCR, human CCR; ES, embryonic stem; KI,
knockin; MPO, myeloperoxidase; ORF, open reading frame; WT, wild type.
The Journal of Immunology
Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00
was prepared by the Pfizer Medicinal Chemistry group, as previously de-
scribed (21). All chemokines were obtained from PeproTech, unless oth-
erwise indicated, checked for purity by HPLC, and verified to be free from
endotoxin using the limulus amebocyte lysate assay (Associates of
Cape Cod).
Animals
DBA/1 mice were obtained from The Jackson Laboratory. All animals
were certified to be free from viral pathogens and were allowed food and
water ad libitum. All experimental protocols were reviewed and approved
by the Pfizer Institutional Animal Care and Use Committee.
Reagents
BSA was purchased from Sigma-Aldrich. Heparin was purchased from
American Pharmaceutical Partners. Dulbecco’s PBS without calcium chlo-
ride and magnesium chloride (PBS), HBSS, and geneticin were obtained
from Invitrogen Life Technologies. FBS was purchased from HyClone.
RPMI 1640, HEPES, glutamine, and penicillin/streptomycin were all ob-
tained from BioWhittaker. Tissue culture medium for cell cultures con-
sisted of RPMI 1640 containing FBS (10%), L-glutamine (2 mM), HEPES
(10 mM), penicillin (100 U/ml), and streptomycin (50
g/ml).
Design of the CCR1 knockin (KI) construct
The CCR1 replacement construct was prepared using genomic DNA from
a mouse (strain 129) DNA library. The hCCR1 open reading frame (ORF)
was subcloned into the expression vector, pcDNA3.1 (Invitrogen Life
Technologies). From this vector, p3MIP34, a hCCR1/Bgh pA fragment,
was excised and spliced into the CCR1 replacement construct 3⬘to the
initiating ATG of the mouse CCR1 (Fig. 1). The CCR1 KI construct com-
prised 4.3-kb 5⬘and 1.0-kb 3⬘homology arms from the mouse genomic
region, flanking both the hCCR1/Bgh pA and the pgk-neo resistance cas-
sette running in the opposite orientation. This construct was subcloned into
pBluescript (Stratagene). For negative selection, the herpes simplex virus
thymidine kinase gene was inserted outside of the 5⬘homology arm in the
opposite orientation. Targeting into mouse embryonic stem (ES) cells by
homologous recombination replaced the mouse ORF of the CCR1 gene
with the hCCR1 ORF.
ES cell transfection and generation of hCCR1 KI mice
The culture procedures for ES cells have been previously described (22).
The CCR1 construct was linearized by digestion at a unique 5⬘NotI site
and electroporated into DBA-252 ES cells (mouse strain DBA1/LacJ (23)).
After 7 days of selection with geneticin at 175
g/ml, surviving ES cell
colonies were picked and expanded for further analysis. Targeting by ho-
mologous recombination was demonstrated by Southern blot analysis. ES
cell clones that had undergone the correct targeting event were karyotyped
and then injected into C57BL/6 blastocyst stage embryos to generate chi-
meric mice. Male chimeras were bred with DBA/1lacJ females transmitting
the targeted mutation through the germline of the offspring produced. The
resulting hCCR1 KI/wild-type (WT) (h/⫹)F
1
males and females were bred
together to produce homozygous hCCR1 KI (h/h) F
2
animals. Animals
were backcrossed onto a DBA/1 background for at least nine generations.
Analysis of mouse CCR1 and hCCR1 expression
Cells were collected from hCCR1 KI and WT animals and analyzed for
hCCR1 expression by mRNA and cell surface receptor expression by
FACS analysis. Neutrophils were collected from the peritoneal cavity 18 h
after injection of 1.0 ml of 6% casein, whereby elicited macrophages were
collected 3– 4 days after casein injection. Lymphocytes were collected
from the spleen or lymph nodes. RNA was isolated using the RNeasy
purification method (Invitrogen Life Technologies) with DNase treatment.
RT-PCR was done by reverse transcription using both random hexamers
and oligo(dT) primers and avian myeloblastosis virus reverse transcriptase
(Roche Diagnostics) and 5
g of RNA. Mouse-specific primers used were:
sense primer, 5⬘-ATGCCAAAAGACTGCTGTA-3⬘hybridizing at bases
69 –87, and antisense primer, 5⬘-GAGGAGGAAGAATAGAAGAGTA-3⬘
hybridizing at bases 732–750. Human specific primers used were: sense
primer, 5⬘-GTGCCAGAAGGTGAACGA hybridizing at bases 69 –87, and
antisense primer, 5⬘-GAGAAAAAAGATGATCATG hybridizing at bases
732–750. Thirty cycles of PCR were performed with an annealing temper-
ature of 55°C using PerkinElmer AmpliTaq polymerase and the following
final concentrations of components: 1
M each primer, 2 mM MgCl
2
,50
M dNTPs, and 3
l of cDNA as template. Cell surface expression of
CCR1 was assessed by FACS analysis using anti-hCCR1 (Lifespan) or the
appropriate isotype control.
Chemotaxis assays
Chemotaxis was conducted in 48-well chemotaxis chambers purchased
from NeuroProbe, as previously described (14). Briefly, agonists were di-
luted in RPMI 1640 containing 0.1% BSA, then added to the bottom wells
of the chamber. A polyvinylpyrrolidone-free filter with 5-
m pores (Neu-
roProbe) was placed between the upper and lower wells of the chamber.
Cells were then added to the top chamber (2 ⫻10
5
) in the presence or
absence of various concentrations of CP-481,715, and the apparatus was
incubated for 60 min in a 5% CO
2
humidified incubator at 37°C. After the
incubation period, the nonmigrating cells were removed from the upper
chamber, and the top of the filter was wiped. The bottom portion of the
filter was stained with Diff-Quik (Dade Behring), and the number of mi-
grating cells in six random fields was enumerated with a microscope.
Whole blood actin polymerization
Mouse blood, collected in EDTA, was incubated with various dilutions of
CP-481,715 or diluent for 5 min at room temperature. CCL3 (10 nM) was
then added, and after 50 s the reaction was terminated by adding FACS
lysing solution (BD Biosciences) containing paraformaldehyde (Electron
Microscopy Sciences). After 10 min, the cells were collected by centrifu-
gation, washed with PBS, and stained for1hatroom temperature in the
FIGURE 1. Engineering the hCCR1 KI gene into ES
cells. The hCCR1 KI construct (A) replaced the endog-
enous mouse CCR1 ORF (B) via homologous recom-
bination. This resulted in a complete KI of the human
ORF while simultaneously deleting mouse CCR1 and
introducing the pgk-neo cassette for positive selection
(C). Upon this event, a unique PstI site from the hCCR1
was introduced. This resulted in a 6.3-kb mutant frag-
ment as opposed to the 9.0-kb WT (D). Abbreviations:
P, PstI; H, HindIII; X, XbaI; R, EcoRI; S, SmaI.
3142 ANTI-INFLAMMATORY ACTIVITY OF CP-481,715 IN TRANSGENIC MICE
dark with a solution containing lysophosphatidylcholine (Sigma-Aldrich),
paraformaldehyde, and nitrobenzoxadiazole phallacidin (Molecular
Probes). The cells were then washed with PBS containing 2% FBS, and the
fluorescence was quantitated using a FACScan (BD Biosciences).
Skin challenge study
Mice were injected intradermally at 0 and 2 h with 1
g of CCL3 or vehicle
(0.5% BSA). After 4 h, skin was excised and frozen. An 8-mm skin punch
was made from the frozen skin, and each section was placed into 1 ml of
50 mM K
2
PO
4
(pH 6.0) buffer. The skin was homogenized, freeze thawed
twice, and centrifuged, and the supernatants were collected for analysis of
myeloperoxidase (MPO) levels.
MPO assay
Test samples were placed into wells of a 96-well flat-bottom plate con-
taining 150
l of substrate (750
lofN,N-dimethylformamide (Sigma-
Aldrich), 49.25 ml of buffer, 10 mg of o-dianisidine (Sigma-Aldrich), and
11
lof3%H
2
O
2
(Sigma-Aldrich)). The plate was incubated at 37°C for
15 min, and the reaction was stopped by adding 100
l of 0.4 M glycine
(pH 10.4). The absorbance was read at 450 nm, and the amount of MPO
was determined from a standard curve.
Air pouch model of cell infiltration
Subcutaneous air pouches were formed on the back of animals, as previ-
ously described (24). Briefly, 3 ml of air was injected s.c. on day 1 and then
reinjected again 3 days later in the same area. On the fourth day, animals
received a single i.p. injection of CP-481,715, followed by two injections
of CCL3 (1
g/ml) administered directly into the air pouch at time 0 and
2 h. The pouches were washed with 3 ml of PBS containing 10 mM EDTA
2 h after the last injection of CCL3. The number of cells was counted
microscopically.
Delayed-type hypersensitivity model
Delayed-type hypersensitivity was assessed in SRBC-sensitized mice.
Briefly, defibrinated SRBC (REMEL) were washed, and 1 ⫻10
6
cells were
injected i.v. into animals to sensitize them. Six days later, mice were in-
jected into the footpad with 10
8
SRBCs in 25
l. Footpad swelling was
measured with calipers 24 h after rechallenge. In some animals,
CP-481,715 was administered as a single injection (i.p.) at the time of
rechallenge. In other studies, CP-481,715 was administered i.p. daily be-
ginning at the time of sensitization.
Cytokine analysis in sensitized animals was determined on splenic lym-
phocytes. Spleens were collected from hCCR1 KI and WT animals 6 days
after sensitization, and a single cell suspension was made. Lymphocytes
were isolated over Ficoll (Sigma-Aldrich), and cells were cultured in the
presence or absence of 0.5 or 0.25
g/ml Con A. The supernatants were
collected for analysis of IL-2 and IFN-
␥
24 h later. IL-2 and IFN-
␥
con-
centrations were determined by ELISA (R&D Systems).
Statistical analysis
Statistical comparisons between groups were performed using Student’s t
test. A p⬍0.05 was considered significant.
Results
Characterization of hCCR1 in KI animals
Gene targeting was confirmed by Southern blot analysis initially in
ES cells and subsequently using DNA from hCCR1 KI animals
(Fig. 2). External probes (5⬘(SE0.8) and 3⬘(EX0.2)) were used in
combination with the appropriate restriction enzyme digests to
yield restriction fragment-length polymorphisms indicative of 9-kb
WT and 6.3-kb hCCR1 KI alleles (Fig. 2, Aand B). Animals were
born with the expected ratios and were viable and healthy. RT-
PCR analysis of neutrophils, macrophages, and lymphocytes from
WT and hCCR1 KI animals revealed that CCR1 was expressed in
all three cell types, with murine CCR1 expressed only in cells from
WT animals and hCCR1 expressed only in cells from hCCR1 KI
animals (Fig. 2C). Cell surface expression of hCCR1 was assessed
by FACS analysis (Fig. 3) and demonstrated that hCCR1 was
present on neutrophils, monocytes, and lymphocytes from hCCR1
KI mice.
Functional responses of cells
To ensure that hCCR1 expressed on leukocytes was functional and
responded to murine CCL3, an essential attribute to use these an-
imals to evaluate the effects of CP-481,715 on inflammatory re-
sponses, we isolated neutrophils from the peritoneal cavity follow-
ing casein elicitation and assessed their ability to migrate in
response to human and murine CCL3 in vitro. As shown in Fig. 4,
cells isolated from hCCR1 KI animals migrate in response to both
human and murine CCL3 at levels comparable to cells isolated
from WT animals. This cross-reactivity of murine CCL3 on
hCCR1 was also confirmed in chemotaxis assays using the human
monocyte cell line THP-1 (data not shown). These results demon-
strate that both murine and human CCL3 are active on hCCR1, and
confirm that hCCR1 expressed on cells from KI animals is
functional.
We next assessed the ability of CP-481,715 to inhibit the che-
motaxis of neutrophils, lymphocytes, and macrophages from
hCCR1 KI and WT mice in response to CCL3. As shown in Fig.
5, Aand B, CP-481,715 inhibited the chemotaxis of neutrophils
and lymphocytes taken from hCCR1 KI mice (but not WT mice)
in response to CCL3, demonstrating the specificity of CP-481,715
for the human receptor and confirming that the CCL3-induced che-
motaxis of cells isolated from hCCR1 KI animals was hCCR1
mediated. As shown in Fig. 5C, the concentration of CP-481,715
necessary to inhibit the chemotaxis of both lymphocytes and
neutrophils (IC
50
⫽114 and 93 nM, respectively) isolated from
hCCR1 KI mice was similar to that necessary to inhibit the
chemotaxis of human monocytes in response to CCL3 (IC
50
⫽
FIGURE 2. Expression of hCCR1 in ES cells and in hCCR1 KI ani-
mals. A, Southern blot of genomic DNA from ES cells that survived G418
selection showing targeted KI of hCCR1 (KI, 6.3 kb) and murine CCR1
(WT, 9 kb). Genomic DNA was restriction digested with PstI, Southern
blotted, and probed with the external 5⬘probe 0.8-kb SE0.8. B, Southern
blot analysis of genomic DNA obtained from the tails of pups born to
heterozygous F
1
matings. Autoradiographs show the 9-kb WT (murine
CCR1) and 6.3-kb KI (human CCR1) bands. ⫹/⫹⫽WT animals; h/⫹⫽
hCCR1 KI/mouse WT heterozygous; h/h ⫽hCCR1 KI homozygous. C,
RT-PCR analysis of hCCR1 and murine CCR1 mRNA in neutrophils iso-
lated from the peritoneal cavity 24 h after casein injection (lanes 1 and 2),
monocytes isolated from the peritoneal cavity 4 days after casein injection
(lanes 3 and 4), and lymphocytes isolated from lymph nodes (lanes 5 and
6) obtained from hCCR1 KI and WT animals.
3143The Journal of Immunology
97 nM). Interestingly, although hCCR1 was expressed on mac-
rophages from KI animals, CP-481,715 only partially inhibited
the chemotactic response (ⱕ25%) to CCL3, suggesting alter-
native receptors other than CCR1 dominate this chemotactic
activity.
Whole blood actin polymerization
To ensure that CP-481,715 would have activity in hCCR1 KI
mouse whole blood, we assessed the ability of CP-481,715 to in-
hibit CCL3-induced actin polymerization in neutrophils by FACS
analysis. As shown in Fig. 6, CCL3-induced neutrophil actin po-
lymerization in hCCR1 KI mouse blood was inhibited by
CP-481,715 with an IC
50
of 33 nM. The concentration of CP-
481,715 necessary to inhibit this response in hCCR1 KI blood was
similar to that previously reported to inhibit monocyte actin poly-
merization in human whole blood in response to CCL3 (IC
50
⫽58
nM) (14). CP-481,715 did not inhibit CCL3-induced actin poly-
merization in blood taken from WT animals.
CP-481,715 inhibits CCL3-induced cell infiltration in hCCR1
KI mice
The ability of CP-481,715 to inhibit in vivo cell migration in
hCCR1 KI animals was clearly demonstrated in two separate mod-
els. In the first model, CP-481,715 inhibited neutrophil infiltration
in response to an intradermal injection of CCL3 as assessed in skin
biopsies by MPO levels (Fig. 7A) with an ED
50
of 0.23 mg/kg. In
the second model, neutrophil infiltration into an air pouch was
inhibited by CP-481,715 with an ED
50
of 0.22 mg/kg (Fig. 7B). In
FIGURE 3. FACS analysis for hCCR1 expression in
cells isolated from hCCR1 KI animals. A, hCCR1 ex-
pression on neutrophils in the peripheral blood of
hCCR1 KI mice. B, hCCR1 expression on peritoneal
macrophages isolated from hCCR1 KI mice 3– 4 days
following casein elicitation. C, hCCR1 expression on
lymphocytes isolated from the lymph nodes of hCCR1
KI mice. Background staining with the isotype control
(Iso) is shown for comparison with staining with a hu-
man specific CCR1 Ab.
3144 ANTI-INFLAMMATORY ACTIVITY OF CP-481,715 IN TRANSGENIC MICE
these models, CCL3 primarily induces a neutrophil infiltration as
assessed microscopically. No inhibition was observed on CCL3-
induced cell infiltration in WT animals. Furthermore, as shown in
Fig. 7C, although CP-481,715 was able to inhibit cell infiltration in
hCCR1 KI animals in response to CCL3, it did not inhibit neutro-
phil infiltration in response to the murine chemokine KC, a neu-
trophil chemotactic agent acting through CXCR2. The plasma lev-
els of CP-481,715 necessary to inhibit 90% of the cell migration in
response to CCL3 using the air pouch model are shown in Fig. 7D,
and suggest that a trough level of 40 ng/ml, maintained for only
2 h, was sufficient to inhibit the inflammatory cascade in this
model. These studies confirm the in vivo selectivity of CP-481,715
and illustrate its ability to inhibit CCL3-dependent cell migration
at clinically achievable dose levels.
Effects of CP-481,715 on delayed-type hypersensitivity
Because lymphocyte chemotaxis in response to CCL3 was also
CCR1 dependent and could be blocked by CP-481,715 using cells
from hCCR1 KI animals, we next examined the ability of CP-481,715
to inhibit inflammation in a more classical lymphocyte-mediated in-
flammatory response. As shown in Fig. 8A, CP-481,715 significantly
inhibited delayed-type hypersensitivity with an ED
50
of 0.88 mg/kg.
This inhibition was observed when CP-481,715 was administered as
a single injection at the time of rechallenge. No inhibition of delayed-
type hypersensitivity with CP-481,715 was observed in WT animals
(data not shown), again confirming the selectivity of CP-481,715 and
the dependence of this response on CCR1. Furthermore, this inhibi-
tion of delayed-type hypersensitivity in hCCR1 KI animals was also
observed when treatment was delayed up to 6 h after rechallenge in
these animals (data not shown).
Effects of CP-481,715 on Th1 cytokine responses
Although CP-481,715 was able to inhibit delayed-type hypersen-
sitivity when administered at the effector stage of the response, we
next wanted to determine whether blockade of CCR1 altered cy-
tokine production in sensitized animals, as previously reported in
CCR1
⫺/⫺
animals using other models (16, 17). As shown in Fig.
8, Band C, the level of both IFN-
␥
and IL-2 was reduced ⬎50%
in supernatants from cells obtained from CP-481,715-treated ani-
mals in response to Con A, suggesting that CP-481,715 altered
general inflammatory responses and T cell activity at the spleen.
Discussion
We describe the generation of a transgenic mouse expressing
hCCR1 in place of murine CCR1 and illustrate its utility to assess
the anti-inflammatory properties of the human specific CCR1 an-
tagonist CP-481,715. CCR1 KI mice express hCCR1 in cell types
comparable to those expressing murine CCR1 in WT animals, and
were demonstrated to be functional both in vitro and in vivo. In
FIGURE 4. Neutrophils isolated from hCCR1 KI and WT animals mi-
grate in response to both human (h) and murine (m) CCL3 at comparable
levels. Neutrophils were isolated from the peritoneal cavity of hCCR1 and
WT animals 24 h after casein injection. Chemotaxis was assessed using a
Boyden chamber with 1.0 and 0.1 nM human and murine CCL3. The data
are representative of three experiments using quadruplicate wells for anal-
ysis and are represented as the mean ⫾SD. No statistical significance was
achieved in comparing the chemotaxis of cells from hCCR1 KI vs WT
animals for a given concentration of CCL3.
FIGURE 5. CP-481,715 inhibits the chemotaxis of cells obtained from
hCCR1 KI mice at concentrations similar to that of human cells. Neutro-
phils and macrophages were isolated from the peritoneal cavity after casein
elicitation, while lymphocytes were isolated from the spleen and lymph
nodes. Cells were analyzed for chemotaxis using a 48-well Boyden cham-
ber with CCL3. A, Effect of CP-481,715 on the chemotaxis of cells ob-
tained from hCCR1 KI or WT mice in response to CCL3. The data are
expressed as the chemotactic index (number of cells migrating in response
to CCL3/number of cells migrating in the absence of CCL3) ⫾SD in the
presence and absence of 25
M CP-481,715. The data are representative of
at least three separate experiments. ⴱ,p⬍0.05. B, The concentration of
CP-481,715 necessary to inhibit neutrophil chemotaxis was assessed in
cells obtained from hCCR1 KI and WT animals. The data are expressed as
the percentage of inhibition of chemotaxis ⫾SD and are representative of
at least three separate experiments. ⴱ,p⬍0.05. C, The concentration of
CP-481,715 required to inhibit chemotaxis was assessed using hCCR1 KI
neutrophils, monocytes, and lymphocytes, and compared with human pe-
ripheral blood monocytes and human THP-1 cells. The data are expressed
as the percentage of inhibition of chemotaxis ⫾SD using a minimum of three
replicates. Significant inhibition (p⬍0.05) by CP-481,715 was obtained for all
cell types, except hCCR1 KI monocytes, in which significance was only
achieved at the upper concentrations of CP-481,715. NS, p⬎0.05. The data
are representative of at least four separate experiments. The chemotactic index
obtained in the absence of CP-481,715 was: 4.4-fold for hCCR1 KI neutro-
phils; 6.0-fold for CCR1 KI lymphocytes; 3.0-fold for hCCR1 KI macro-
phages; 2.8-fold for human monocytes; and 4.6-fold for THP-1 cells.
3145The Journal of Immunology
addition, the concentration of CP-481,715 necessary to inhibit
CCL3-induced chemotaxis or whole blood actin polymerization
using cells from these animals was similar to those concentrations
necessary to inhibit responses using human cells. As such, studies
in hCCR1 KI animals should be useful to demonstrate the anti-
inflammatory properties of CP-481,715 and help predict the
plasma levels necessary to inhibit cell migration in clinic.
The functional expression of hCCR1 in these animals allowed
us to assess the role of CCR1 in several models of inflammation.
Blockade of hCCR1 with CP-481,715 inhibited neutrophil infil-
tration induced by CCL3 and prevented inflammatory responses in
a model of delayed-type hypersensitivity. Furthermore, in agree-
ment with data generated in CCR1
⫺/⫺
animals, CP-481,715 also
modulated Th1 cytokine responses in immunized animals (16).
The CCR1 dependence of these responses and the selectivity of
CP-481,715 for hCCR1 were clearly demonstrated by the lack of
effect of CP-481,715 in any of these models using WT animals that
express murine CCR1. Collectively, these studies clearly illustrate
the anti-inflammatory potential of a CCR1 antagonist.
The plasma trough level of CP-481,715 necessary to inhibit both
delayed-type hypersensitivity and CCL3-induced cell infiltration at
the 90% efficacy level was 40 ng/ml (achieved with a 1.0 mg/kg
dose level). Interestingly, it was not necessary to continuously
maintain these levels to observe activity. In fact, maintaining
plasma levels for a 24-h period in the delayed-type hypersensitivity
model by multiple injections did not result in improved efficacy
(data not shown) as compared with a single injection at the time of
rechallenge in which plasma levels were only detectable for only
a few hours. One explanation for this might relate to interrupting
FIGURE 7. CP-481,715 inhibits cell infiltration in response to CCL3, but not KC in hCCR1 KI animals. A, Cell infiltration into skin following an
intradermal injection of 1
g of CCL3, as assessed by MPO levels in excised skin punches. The data represent the mean MPO level/ml in an 8-mm skin
punch homogenized in 1.0 ml of buffer from a minimum of five animals per group. The data are representative of three separate experiments. ⴱ,p⬍0.05.
B, Neutrophil infiltration into an air pouch following two injections of 1
g of CCL3. The number of cells migrating into the air pouch was 3.6 ⫻10
6
in
response to CCL3. The data represent the percentage of inhibition of this cell infiltration ⫾SD by various dose levels of CP-481,715 using a minimum
of five animals per group. The data are representative of greater than 10 separate experiments. ⴱ,p⬍0.05. C, Neutrophil infiltration into an air pouch
following two injections of either KC or CCL3 and the level of inhibition by a 10 mg/kg dose of CP-481,715. The mean cell number per pouch ⫾SD from
a minimum of five animals per group is shown. The data are representative of three separate experiments. ⴱ,p⬍0.05. D, Serum levels of CP-481,715
following a 1.0 mg/kg dose (i.p.) associated with the inhibition of cell infiltration into air pouches in response to CCL3.
FIGURE 6. CP-481,715 inhibits CCL3-induced actin polymerization in
whole blood taken from hCCR1 KI mice, but not WT mice. Blood was
collected in EDTA from either hCCR1 KI or WT animals. CP-481,715 was
added to the blood, followed by the addition of 10 nM CCL3. Actin po-
lymerization was determined in the neutrophil population by FACS anal-
ysis. The data represent the percentage of inhibition of the median channel
fluorescence induced by CCL3 ⫾SD from triplicate samples (median
channel fluorescence ⫽150 for CCL3 in the absence of CP-481,715). The
data are representative of four separate experiments. ⴱ,p⬍0.05.
3146 ANTI-INFLAMMATORY ACTIVITY OF CP-481,715 IN TRANSGENIC MICE
the inflammatory cascade, whereby cells migrating into the site of
inflammation normally become activated and secrete additional
chemokines, including CCL3, thus recruiting additional cells.
Once this cascade is disrupted through CCR1 inhibition, it results
in long-term anti-inflammatory effects, as suggested by this study.
In fact, in clinical trials conducted in rheumatoid arthritis patients,
CP-481,715 was able to significantly decrease cell infiltration (25)
at plasma trough levels comparable to those achieved in these stud-
ies. Consequently, hCCR1 KI animals are useful to help predict the
efficacious dose levels necessary to see activity in clinic.
Although studies in hCCR1 KI animals are valuable to help
assess the ability of a human specific CCR1 antagonist to prevent
cell infiltration, extending these studies in animal disease models
to help predict human disease indications must be done with cau-
tion because the dominant role of CCR1 in CCL3-induced che-
motactic responses varies with cell type between human and
mouse. As with human cells, the lymphocyte chemotactic re-
sponses induced by CCL3 in hCCR1 KI mice can be blocked by
CP-481,715. This is not the case for monocytes. Whereas human
monocyte chemotaxis in response to CCL3 was completely inhib-
ited by CP-481,715 (14), monocytes taken from hCCR1 KI mice
were only partially inhibited. Studies using CCR1
⫺/⫺
mice have
also indicated that CCR1 is important for neutrophil and lympho-
cyte migration, but not monocyte migration, as indicated by the
lack of effects on monocyte infiltration into the peritoneal cavity in
response to thioglycolate (16). Although this difference between
mouse and human is potentially related to differences in CCR1
receptor expression on these cells, macrophages from mice did
express high levels of CCR1. An alternative explanation is that
specific differences in the expression of other chemokine receptors
are responsible for the decreased role of CCR1 on monocytes in
mice. A likely candidate is CCR5, which also uses CCL3 as a
ligand. CCR5
⫺/⫺
animals have been shown to have a defect in
monocyte migration in response to thioglycolate elicitation (26)
(unlike CCR1
⫺/⫺
mice (16)), suggesting that CCR5 may serve a
more important and dominant role on monocyte migration in the
mouse. As such, it is possible that CCR5 may be the dominant
receptor for CCL3 on monocytes in mice, which is in contrast to
what we have observed on human monocytes (14).
Another important difference in CCR1 function between mice
and humans that we observed in our studies is its role on neutrophil
migration. In mice, CCR1 is an important neutrophil chemotactic
factor, as illustrated both in vitro and in vivo by our studies and
further supported by studies in CCR1
⫺/⫺
mice in which neutrophil
infiltration was suppressed in response to thioglycolate (16). In
contrast, the role of CCR1 in human neutrophil responses has been
controversial. For example, some reports have indicated that neu-
trophils isolated from human peripheral blood require stimulation
by cytokines such as GM-CSF to express CCR1 (27), while others
have claimed CCR1 is expressed on neutrophils, but the functional
response is limited to certain CCR1 ligands such as leukotactin-1
(28). Interestingly, when CCL3 is injected intradermally into nor-
mal human subjects, a robust neutrophil infiltration was observed
as early as 2 h after injection (8), raising questions as to whether
isolation techniques used for human cells might down-regulate
CCR1 expression and/or alter ligand-induced functional responses.
Nonetheless, differences exist between humans and mice that have
to be considered when assessing the disease potential of a CCR1
antagonist in which neutrophils are involved in the pathogenesis.
Studies to address the role of CCR1 using pharmacological
agents in mice have been limited due to the human specificity of
most agents. One exception is the CCR1 antagonist BX-471,
which has been used to demonstrate activity in several animal
disease models, including transplant rejection, renal fibrosis, ar-
thritis, and multiple sclerosis (29). Although these studies support
a role for CCR1 in modulating inflammation, a limitation is the
high concentration of the agent needed to inhibit rodent CCR1
(⬃100-fold higher than that necessary for hCCR1) (30). As such,
one concern is that at these high dose levels, other rodent G pro-
tein-coupled receptors (including chemokine receptors) may also
have been inhibited, a selectivity issue that is difficult to address
without cloning receptors and evaluating the compound at these
concentrations on a series of rodent G protein-coupled receptors.
More recently, a series of CCR1 antagonists have been described
FIGURE 8. CP-481,715 inhibits delayed-type hypersensitivity and de-
creases Th1 cytokine production by isolated spleen cells. A, hCCR1 KI
mice were injected i.v. with SRBC, then challenged 5 days later into the
footpad with SRBC. Various dose levels of CP-481,715 were administered
i.p. at the time of rechallenge. The data represent the mean footpad swell-
ing ⫾SD at 24 h after rechallenge from a minimum of five animals per
group. The data are representative of at least 10 separate experiments. ⴱ,
p⬍0.05. B, IL-2 production by spleen cells stimulated with Con A from
SRBC-sensitized mice treated with 1.0 mg/kg CP-481,715. The data are
expressed as the mean concentration of IL-2 in supernatants ⫾SD from a
minimum of three replicates. The data are representative of three separate
experiments. ⴱ,p⬍0.05. C, IFN-
␥
production by spleen cells stimulated
with Con A from SRBC-sensitized mice treated with 1.0 mg/kg
CP-481,715. The data are expressed as the mean concentration of IFN-
␥
in
supernatants ⫾SD from a minimum of three replicates. The data are rep-
resentative of three separate experiments. ⴱ,p⬍0.05.
3147The Journal of Immunology
that have equipotent activity on murine CCR1 and hCCR1 (31).
Although these antagonists have demonstrated activity in a murine
arthritis model, again selectivity vs other murine receptors was not
reported. Nonetheless, our studies do support a role for CCR1 on
neutrophil- and lymphocyte-mediated inflammatory responses in
mice, suggesting that CCR1 may play a role in models of trans-
plant rejection and multiple sclerosis, as suggested with BX-471.
Our studies do raise questions, however, on any effects observed
with these agents on monocyte infiltration, as this is unlikely to be
directly CCR1 mediated.
Generation of human chemokine receptor KI animals represents
a viable strategy to assess the in vivo activity of human specific
chemokine receptor antagonists. Leukocytes from KI animals ex-
press hCCR1 and migrate to CCR1 ligands. Studies in hCCR1 KI
animals demonstrate the potent ability of CP-481,715 to decrease
CCL3-induced cell infiltration, prevent inflammatory responses
(delayed-type hypersensitivity), and alter cytokine responses in
sensitized animals. These studies underscore the importance of
CCR1 in inflammation and the role of chemokines in these re-
sponses, and raise the possibility that inhibiting CCR1 will mod-
ulate inflammatory responses in clinic. In addition, the ability of
CP-481,715 to inhibit cell infiltration at dose levels and plasma
concentrations achievable in clinic suggests the potential clinical
utility of this agent in human inflammatory diseases.
Disclosures
All of the authors are employees of Pfizer Global Research and
Development.
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3148 ANTI-INFLAMMATORY ACTIVITY OF CP-481,715 IN TRANSGENIC MICE
