By
From the Department of * Oncology and Experimental Pathology, Bristol-Myers Squibb
Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000; and the § Department of
Microbiology, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Connecticut
06492-7660
Chemokines are a structurally related family of cytokines that are important for leukocyte trafficking. The C-C chemokine monocyte chemoattractant protein-1 (MCP-1) is a potent
monocyte activator in vitro and has been associated with monocytic infiltration in several inflammatory diseases. One C-C chemokine receptor, CCR2, has been identified that mediates
in vitro responses to MCP-1 and its close structural homologues. CCR2 has also recently been
demonstrated to be a fusion cofactor for several HIV isolates. To investigate the normal physiological function of CCR2, we generated mice with a targeted disruption of the ccr2 gene.
Mice deficient for CCR2 developed normally and had no hematopoietic abnormalities. However, ccr2/
mice failed to recruit macrophages in an experimental peritoneal inflammation
model. In addition, these mice were unable to clear infection by the intracellular bacteria, Listeria monocytogenes. These results suggest that CCR2 has a nonredundant role as a major mediator
of macrophage recruitment and host defense against bacterial pathogens and that MCP-1 and
other CCR2 ligands are effectors of those functions.
Chemokines are a large family of structurally related secreted proteins that are important for leukocyte trafficking (1). The regulated interaction of chemokines with
their respective receptors are thought to mediate the controlled recruitment of specific leukocyte subpopulations required during host defense and inflammation (4). The specific biological functions of chemokines and their receptors
has been difficult to predict, since most chemokines bind
more than one receptor and most chemokine receptors bind
more than one chemokine ligand in vitro. The analysis of
mutant mice lacking a single chemokine ligand or chemokine receptor gene has been useful for determining some of
their specific physiological functions. Nonredundant roles
in neutrophil and eosinophil recruitment (5), hematopoiesis (5), inflammatory response to viral infection (9),
and neutrophil-mediated host defense, granuloma formation, and cytokine balance (6) have been demonstrated in
mice lacking the chemokine receptors CXCR2 and CCR1
and the chemokine ligands macrophage inflammatory protein (MIP) -1 The chemokine ligand monocyte chemoattractant protein-1 (MCP-1) is a potent in vitro monocyte activator that
has been associated with monocytic infiltration in several
inflammatory diseases (10). Two related human receptors,
CCR2A and CCR2B, have been identified that mediate in
vitro responses to MCP-1, and one homologous murine receptor, CCR2, has been identified that mediates in vitro
responses to the murine MCP-1 analogue, JE (11). Both
human and murine CCR2 function as receptors for several
other close structural homologues of MCP-1 (13), and
human CCR2 can also function as a fusion coreceptor for
several HIV isolates (18, 19). Transgenic mouse models
have demonstrated monocyte/macrophage recruitment to
sites of human MCP-1 or murine JE expression (20), and
neutralizing antibody studies have implicated MCP-1 as a
major mediator of macrophage recruitment in several inflammatory models (23). These studies have suggested
that MCP-1 is important for monocyte/macrophage recruitment in vivo, and that CCR2 may mediate such in
vivo responses. To determine if CCR2 may play a role in
macrophage recruitment and function, we have generated
mice with a targeted disruption of the ccr2 gene. Our studies indicate that CCR2 has a nonredundant function as a
major mediator of macrophage recruitment and host defense to bacterial infection.
Targeted Disruption of the Murine ccr2 Gene.
The murine ccr2
gene was cloned from a 129/Sv embryonic stem cell genomic library using a 150-bp ccr2 cDNA fragment as probe. A 1.1-kb
XbaI-BamHI fragment containing part of the 3 Antibodies.
The region of ccr2 encoding the 49 amino-terminal
amino acids was amplified by PCR using the primers: 5 Flow Cytometry and Immunoprecipitations.
Flow cytometry was
performed with a flow cytometer (Coulter Epics Profile II;
Coulter Corp., Hialeah, FL). Single-cell suspensions were prepared
from femoral bone marrow and stained as previously described
(27). 5 × 105 cells were first incubated with 1 µg of anti-CCR2
IgG and then incubated with PE-conjugated anti-rabbit IgG and
FITC-F4/80. For immunoprecipitations, 6 × 106 bone marrow
cells were labeled overnight with 1 mCi [35S]methionine. Cell
extracts were prepared and immunoprecipitation performed as
previously described (26), using 1 µg of anti-CCR2 IgG.
Thioglycollate-induced Peritonitis and Peritoneal Leukocyte Counts.
Peritoneal leukocytes were lavaged with 6 ml of RPMI1640/10%
fetal bovine serum and total cell counts determined with a hemacytometer. 5 × 104 cells were cytospun, stained with Diff-Quik,
and the percentage of macrophages, neutrophils, and eosinophils
determined from a count of at least 300 cells. These percentages
were multiplied by total cell number to obtain the number of peritoneal macrophages, neutrophils, and eosinophils. Thioglycollate-elicited cells were obtained by peritoneal lavage 72 h after i.p. injection of 2 ml of 3% Brewer's thioglycollate broth (Difco, Detroit,
MI) in PBS.
Listeria Monocytogenes Infection.
Listeria monocytogenes (laboratory strain A25616) cultures were grown in Tryptic Soy Broth
(GIBCO BRL, Gaithersburg, MD). Adult mice were infected by
intravenous injection (via tail vein) with 2,500 CFU of L. monocytogenes. The lungs, liver, and spleen of infected animals were removed 5 d after injection or at autopsy for animals that died on
day 4. The numbers of viable L. monocytogenes present in the liver,
spleen, and lungs were determined by plating serial dilutions of
organ homogenates on 5% sheep blood agar. A portion of the
sampled organs were fixed by immersion in 10% neutral-buffered
formalin for histopathological evaluation. Tissues were processed
by routine methods, embedded in paraffin, and 5-µm sections
were stained with either hematoxylin and eosin or Gram stain.
The murine ccr2 gene was
inactivated by homologous recombination using 129/Sv-derived embryonic stem (ES) cells (Fig. 1 a). Correctly targeted ES cells were used to generate chimeras, two of
which transmitted the mutant allele to their offspring. Heterozygous mice were intercrossed under pathogen-free conditions, and resulting litters were healthy and normal in size.
Genotypic analysis of such intercrosses indicated that wild-type (+/+), heterozygous (+/
Since ccr2 mRNA is detected in mononuclear cells (11),
CCR2 protein expression was evaluated in bone marrow
cells using a CCR2 polyclonal antibody. Flow cytometry
analysis demonstrated CCR2 costaining with the macrophage
marker F4/80 on bone marrow cells from ccr2+/+ mice
(Fig. 1 c). The same F4/80-positive population was present in bone marrow cells from ccr2 MCP-1, a high-affinity ligand for CCR2, has been associated with monocytic infiltrates in several inflammatory diseases (10), and transgenic mouse models have demonstrated
that monocytes and macrophages are recruited to sites of
MCP-1 expression (20). These results suggested that
CCR2 may be involved in monocyte trafficking during
inflammation. To assess the role of CCR2 in macrophage
recruitment in response to a nonspecific inflammatory stimulus, thioglycollate was injected into the peritoneum of wild-type and homozygous mutant mice. There were no differences in the total number of cells and macrophages in the
peritoneum of uninjected ccr2+/+ and ccr2
Since ccr2
Our results demonstrate that CCR2 is a key mediator of
inflammatory and immune responses and that other chemokine receptors, although functionally redundant with CCR2
in vitro, cannot substitute for CCR2 function in vivo. The
failure of CCR2-deficient mice to recruit macrophages in
response to intraperitoneal thioglycollate injection is the first
genetic evidence for the requirement of a chemokine receptor in macrophage trafficking and identifies CCR2 as a
potential therapeutic target for inflammatory diseases. Neutralizing antibody studies have implicated MCP-1 as a major mediator of macrophage recruitment in several inflammatory models (23), and the ccr2, stromal cell-derived factor-1 (SDF-1), and
eotaxin. Although many chemokines including MIP-1
are
chemotactic for monocytes in vitro and several chemokine
receptors including CCR1 are expressed on monocytes and
macrophages, no chemokines or receptors have been identified so far with a specific role in monocyte or macrophage
function.
-UTR of ccr2 was
inserted between the neomycin resistance cassette (PGK-neo) and
herpes simplex virus thymidine kinase cassette (PGK-TK) of the pPNT vector. A 5.5-kb SpeI-BamHI fragment containing the
amino-terminal coding region of ccr2 was then cloned into the
opposite side of the PGK-neo cassette. A 4.2-kb SalI-XhoI fragment containing the lacZ gene was inserted 3
of the 5.5-kb genomic fragment to encode an in-frame fusion of the 57 NH2-terminal residues of CCR2 and
-galactosidase. The resulting 18.4-kb
targeting vector (pPNT-ccr2lacZ) was linearized with NotI and
electroporated (240V/500 µF) into 129/Sv-derived CJ7 embryonic stem cells. After positive selection with G418 (375 µg/ml)
and negative selection with FIAU, doubly resistant clones were
screened for homologous recombination by Southern blotting
with a 0.5-kb BamHI-SpeI external probe. Homologous recombination was detected at a frequency of 1 in 480 clones, and correctly targeted ES cells were injected into blastocysts or aggregated with morula from ICR mice. Male chimeras derived from
both injection and aggregation were mated with ICR females to
obtain germline transmission of the mutated allele.
-GCGGGAATTCGATGGAAGACAATAATATGTTACCT-3
and 5
-GTAGGGATCCCTAACTGGTTTTATGACAAGGCTCACC-3
. The
amplified fragment was digested with EcoRI and BamHI, cloned
into the EcoRI/BamHI sites of the MS2 polymerase fusion vector pEX34c, and the fusion protein induced as described (26).
The fusion protein was purified from MetaPhor agarose (FMC
Corp., Rockland, ME) gels according to the manufacturer's instructions, and the purified protein injected into New Zealand
white rabbits. IgG was purified from rabbit serum by MabTrapG
(Pharmacia Biotech Inc., Piscataway, NJ).
Generation of ccr2 Null Mice.
), and mutant (
/
) mice
were born at the expected Mendelian ratio (Fig. 1 b).
CCR2-deficient mice bred normally and were histopathologically unremarkable (data not shown).
Fig. 1.
Targeted disruption of the mouse ccr2 gene. (a) The wild-type ccr2 genomic DNA locus is depicted in the middle. Relevant restriction endonuclease sites are indicated: B, BamHI; S, SpeI; X, XbaI. S is a
polymorphic SpeI site present in ICR and absent in 129/Sv DNA. The
targeting vector pPNT-ccr2lacZ is shown at the top. Thick lines represent genomic sequences, and the thin lines represent plasmid DNA sequences. The black box indicates the ccr2 coding exon. The lacZ-, PGK-neo, and PGK-TK cassettes are shown as open boxes. The targeted allele
created by homologous recombination of the targeting vector with wild-type genomic DNA is shown at the bottom. The 0.5-kb SpeI-BamHI genomic fragment used for Southern blot analyses is indicated along with
the lengths of diagnostic restriction fragments. (b) Analysis of offspring
from ccr2+/ heterozygote intercrosses. Tail DNA was digested with SpeI
and analyzed by Southern blotting. The 9-kb fragment indicates a wild-type 129/Sv allele, the 5-kb fragment indicates a wild type ICR allele, and the 3-kb fragment is specific for the recombined allele. (c) Flow cytometry analysis of femoral bone marrow cells isolated from wild-type
(+/+) and mutant (
/
) mice. Cells were stained with anti-CCR2 IgG
and FITC-F4/80 followed by PE-goat anti-rabbit IgG. The rectangle
highlights a CCR2-staining cell population present in wild-type but not
mutant mice. (d) Immunoprecipitation analysis of CCR2 expression in
wild-type (+/+), heterozygous (+/
), and homozygous (
/
) mutant
mice. Labeled protein extracts from femoral bone marrow cells was immunoprecipitated with anti-CCR2 IgG.
[View Larger Version of this Image (65K GIF file)]
/
mice but surface expression of CCR2 was not detected. To confirm the lack of
CCR2 protein, radiolabeled bone marrow cell extracts were
immunoprecipitated with anti-CCR2. A 40-kD protein corresponding to CCR2 was detected in extracts from both
ccr2+/+ and ccr2+/
mice but not from ccr2
/
mice (Fig. 1 d).
The presence of similar F4/80-positive populations in the
bone marrow of wild-type and homozygous mutant mice
suggests that CCR2 is not essential for development of the
mononuclear cell lineage. Flow cytometry of thymocytes,
splenocytes, lymph node, and bone marrow cells with macrophage, T cell, and B cell markers also revealed no differences between ccr2+/+ and ccr2
/
mice (data not shown).
Thus, unlike mice deficient for the chemokine ligands SDF-1
(8) and eotaxin (7) or the chemokine receptors CXCR2
(5) and CCR1 (6), CCR2-deficient mice have no obvious defect in hematopoietic development.
/
mice (Fig. 2).
At 72 h after thioglycollate injection, there were significantly fewer cells recovered from the peritoneum of ccr2
/
mice compared with ccr2+/+ mice (
/
, 7.8 ± 0.8 × 106
cells, mean ± SEM, n = 11; +/+, 15.2 ± 2.3 × 106 cells,
mean ± SEM, n = 11; P <0.01). The number of macrophages in the peritoneal exudate of CCR2-deficient
mice was only one-eighth that of wild-type mice (
/
,
1.4 ± 0.2 × 106 macrophages, n = 11; +/+, 11.4 ± 1.7 × 106 macrophages, mean ± SEM, n = 11; P <0.00005).
Since there was no increase in the number of peritoneal
macrophages in the ccr2
/
mice after thioglycollate injection the data indicates that CCR2 is essential for new macrophage recruitment in response to this non-specific inflammatory stimulus. The recruitment defect was specific to
macrophages, as neutrophil and eosinophil infiltration was
not impaired in mutant animals. Interestingly, there were a significantly greater number of recruited eosinophils in
ccr2
/
mice compared with ccr2+/+ mice. This increase
may represent an enhanced eosinophil recruitment in response to a defect in macrophage recruitment. The inability of CCR2-deficient mice to recruit macrophages in response to thioglycollate suggests that CCR2 and its ligands
are specific mediators of macrophage trafficking during inflammation.
Fig. 2.
Macrophage recruitment defect in ccr2/
mice. Resident and
thioglycollate-elicited (72 h post-i.p. injection) peritoneal cells were lavaged from groups of wild-type (open bars) and ccr2
/
(black bars) mice.
Macrophage (Mac), eosinophil (Eos), and neutrophil (Neut) cell numbers
were determined from total cell numbers after differential staining with Diff-Quik. For resident cell counts, values represent mean ± SEM of 5 wild-type or ccr2
/
mice. For elicited cell counts, values represent mean ± SEM
of 11 wild-type or ccr2
/
mice. * P <0.05, ** P <0.006, ***P <0.00001;
probabilities were determined by the unpaired Student's t-test.
[View Larger Version of this Image (15K GIF file)]
/
mice are defective in the recruitment of new macrophages to the peritoneum, we determined if macrophage-dependent immunity to intracellular
pathogens was also compromised in these mice. During the
early phase of infection with the bacterial pathogen L. monocytogenes, the accumulation of blood-derived macrophages to the foci of infection are critical for controlling
bacterial growth in infected organs (28). Groups of wild-type
and homozygous mutant mice were challenged intravenously with 2,500 CFU of L. monocytogenes. After 5 d, when
macrophages are essential for control of L. monocytogenes infection, all ccr2+/+ mice appeared active and healthy. In
contrast, 33% of ccr2
/
mice died 4 d after infection and
the remainder were moribund at 5 d. The bacterial burdens
in the spleen, liver, and lungs were determined at day 5 (or
at autopsy for ccr2
/
mice that died on day 4), and mutant
mice had between 3-7 log-fold higher listerial titers than
wild-type animals in all tissues (Fig. 3), confirming that
CCR2 is required for listerial clearance. The level of sensitivity of CCR2-deficient mice to L. monocytogenes was similar to that seen in mice lacking the 55-kD tumor necrosis factor receptor (29, 30) or the interferon-
receptor (31). At autopsy, livers and spleens from wild-type animals were
of normal color but enlarged, while livers and spleens from
mutant mice were of normal size but appeared mottled and
off color. Histopathological analyses revealed minimal inflammatory foci comprised of macrophages, neutrophils, and
individual necrotic hepatocytes in the livers of wild-type
mice (Fig. 4, a and c). In contrast, severe, multifocal inflammation and necrosis were observed in the livers of ccr2
/
mice (Fig. 4, b and d). These lesions were comprised of a
central core of coagulative necrosis containing neutrophils
and cellular debris and a rim of degenerating and necrotic
hepatocytes containing abundant intracellular bacteria. Severe, multifocal inflammation and necrosis was also observed in the spleens of ccr2
/
but not wild-type mice (data
not shown).
Fig. 3.
CCR2-deficient
mice cannot clear Listeria infection. Wild-type (open bars) and
ccr2/
(black bars) mice were injected intravenously with 2,500 CFU of L. monocytogenes. Listerial titers in liver, spleen, and
lung were determined from mice
killed 5 d after infection or at autopsy for mice that died after 4 d.
Values represent mean ± SEM
of 15 wild-type or ccr2
/
mice.
For all organs, the difference between wild-type and mutant mice is significant (P <0.0002 by Mann-Whitney two sample nonparametric analysis).
[View Larger Version of this Image (18K GIF file)]
Fig. 4.
Histopathology of
Liver from Listeria-infected mice.
(a) Minimal, focal inflammation in
a wild-type ccr2+/+ mouse (arrow).
(b) Severe, multifocal inflammation and necrosis in a ccr2/
mouse (arrows). (c) Higher magnification of a. The localized inflammatory focus contains macrophages
and neutrophils with necrosis of
individual hepatocytes (arrow). (d)
Higher magnification of b. The lesion is characterized by a central
core of coagulative necrosis containing neutrophils and cellular debris. A rim of degenerating and
necrotic hepatocytes that contain
abundant intracellular Gram-positive bacteria are located at the peripheral margin of the lesion. Insets
are sections stained with Gram stain.
Bars: (a, b) 200 µm; (c, d) 20 µm.
[View Larger Version of this Image (203K GIF file)]
/
mice will provide a
useful genetic system to test the potential role of MCP-1/
CCR2 interactions in these models. The extreme sensitivity of CCR2-deficient mice to L. monocytogenes suggests that
CCR2-mediated responses can be as important for host resistance as TNF-
receptor- and IFN-
receptor-mediated
responses. Paradoxically, transgenic mice expressing high
serum levels of MCP-1 also have increased sensitivity to L. monocytogenes (32). We hypothesize that high levels of
MCP-1 may have partially desensitized CCR2 on blood
monocytes in these transgenic mice, causing a similar, but
less severe, phenotype than in the CCR2-deficient animals. Genetic linkage between sensitivity to L. monocytogenes and
low thioglycollate-induced peritoneal macrophage recruitment has previously been identified in several inbred mouse
strains (33). The identification of the same defects in the
ccr2
/
mice raises the possibility that CCR2 function may
be impaired in some of those strains. It is unclear from these
studies if defects in ccr2
/
mice result specifically from the
lack of MCP-1 signaling, since other ligands (MCP-2, 3, 4, and 5) for CCR2 have been identified (13). Targeted
disruption of each of the CCR2 ligands will be informative
for determining which ligands contribute to the macrophage recruitment and host defense functions mediated by CCR2.
Address correspondence to Rodrigo Bravo, Department of Oncology Bristol-Myers Squibb Pharmaceutical Research Institute P.O. Box 4000, Princeton, New Jersey 08543-4000. Phone: (609) 252-5744; Fax: (609) 252-6051.
Received for publication 7 August 1997.
We thank Ken Class for flow cytometry, Sergio Lira and Alice Lee for blastocyst injections, Anne Lewin for histology, and Cheryl Rizzo for cell culture assistance. We also thank Mark Kowala for valuable discussions and the staff of Veterinary Sciences at Bristol-Myers Squibb for their excellent support.
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