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

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.

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.

Figure 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.

Figure 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).

Figure 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.