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Defects in macrophage recruitment and host defense in mice lacking the CCR2 chemokine receptor.

Kurihara T, Warr G, Loy J, Bravo R - J. Exp. Med. (1997)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000, USA.

ABSTRACT
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.

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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.
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Figure 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.

Mentions: 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 (+/−), 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).


Defects in macrophage recruitment and host defense in mice lacking the CCR2 chemokine receptor.

Kurihara T, Warr G, Loy J, Bravo R - J. Exp. Med. (1997)

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.
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Related In: Results  -  Collection

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Figure 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.
Mentions: 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 (+/−), 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).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus