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. 1998 Feb 16;187(4):601-8.
doi: 10.1084/jem.187.4.601.

Abnormalities in monocyte recruitment and cytokine expression in monocyte chemoattractant protein 1-deficient mice

B Lu  1 , B J Rutledge, L Gu, J Fiorillo, N W Lukacs, S L Kunkel, R North, C Gerard, B J Rollins
Affiliations

Abnormalities in monocyte recruitment and cytokine expression in monocyte chemoattractant protein 1-deficient mice

B Lu et al. J Exp Med. .

Abstract

Monocyte chemoattractant protein 1 (MCP-1) is a CC chemokine that attracts monocytes, memory T lymphocytes, and natural killer cells. Because other chemokines have similar target cell specificities and because CCR2, a cloned MCP-1 receptor, binds other ligands, it has been uncertain whether MCP-1 plays a unique role in recruiting mononuclear cells in vivo. To address this question, we disrupted SCYA2 (the gene encoding MCP-1) and tested MCP-1-deficient mice in models of inflammation. Despite normal numbers of circulating leukocytes and resident macrophages, MCP-1(-/-) mice were specifically unable to recruit monocytes 72 h after intraperitoneal thioglycollate administration. Similarly, accumulation of F4/80+ monocytes in delayed-type hypersensitivity lesions was impaired, although the swelling response was normal. Development of secondary pulmonary granulomata in response to Schistosoma mansoni eggs was blunted in MCP-1(-/-) mice, as was expression of IL-4, IL-5, and interferon gamma in splenocytes. In contrast, MCP-1(-/-) mice were indistinguishable from wild-type mice in their ability to clear Mycobacterium tuberculosis. Our data indicate that MCP-1 is uniquely essential for monocyte recruitment in several inflammatory models in vivo and influences expression of cytokines related to T helper responses.

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Figures

Figure 1
Figure 1
(A i) Wild-type SCYA2 locus. The three exons encoding MCP-1 are shown as hatched boxes; the positions of BamHI and SstI restriction endonuclease sites are indicated in relation to the 5′ BamHI site; the NaeI–HpaI fragment used as a probe in Southern blotting is indicated as the open box. (ii) Targeting construct indicating the transcriptional orientation of the PGK-neo cassette inserted in the second exon of MCP-1. Asterisk denotes the site of an in-frame stop codon engineered in the first exon (see Materials and Methods). (iii) Disrupted allele. (B) Southern blot analysis of wild-type and MCP-1–deficient mice. DNA was extracted from tails of wild-type mice (+/+), and mice heterozygous (+/−) or homozygous (−/−) for the disrupted MCP-1 allele. DNA was digested using SstI and analyzed by Southern blotting using the probe indicated in A. (C) Chemokine expression in wild-type and MCP-1–deficient mice. Peritoneal macrophages from wild-type mice (+/+), and mice heterozygous (+/−) or homozygous (−/−) for the disrupted MCP-1 allele were treated with LPS and radiolabeled using [35S]methionine as described in Materials and Methods. Conditioned medium was analyzed by immune precipitation using anti–MCP-1 antiserum and the precipitates were analyzed by SDS-PAGE (left gel). The supernatants from the anti–MCP-1 precipitation were then subjected to immune precipitation using anti– murine GRO-α/KC, and these precipitates were analyzed by SDS-PAGE (middle gel). In a separate experiment, conditioned medium was analyzed by immune precipitation using anti–murine MCP-3 antiserum (right gel). Arrow indicates position of MCP-3. A small amount of cross-reactivity with MCP-1 can be discerned at ∼30 kD in supernatants from wild-type macrophages.
Figure 2
Figure 2
Thioglycollate elicitation in wild-type and MCP-1–deficient mice. Total and differential leukocyte counts were obtained on the resident peritoneal cells of six wild-type and six MCP-1−/− mice. An additional six wild-type and six MCP-1−/− mice were challenged with intraperitoneal thioglycollate. After 72 h, elicited cells were collected by lavage and analyzed for total cell and differential leukocyte count. Essentially no lymphocytes or mast cells were seen in either genotype. R, resident cells; E, elicited cells. Error bars indicate SEM. Differences in numbers of elicited neutrophils and eosinophils between wild-type and MCP-1−/− mice were not statistically significant by Student's t test.
Figure 3
Figure 3
DTH responses in wild-type and MCP-1–deficient mice. (A) Contact hypersensitivity. Six wild-type and six MCP-1−/− mice were sensitized with DNFB as described in Materials and Methods. 6 d later, sensitized mice as well as six naive wild-type and six naive MCP-1−/− mice were challenged by application of DNFB to one ear. 24 h later, the difference in thickness between treated and untreated ears was determined. C, control naive mice; S, sensitized mice; +/+, wild-type; −/−, MCP-1−/−. Error bars indicate SEM. The difference in ear swelling between sensitized wild-type and sensitized MCP-1−/− mice was not statistically significant. Similar results were observed in males and females. (B) Tuberculin-type hypersensitivity. 10 wild-type and 11 MCP-1−/− mice were sensitized with NP-O-Su as described in Materials and Methods. 7 d later, sensitized mice as well as nine naive wild-type and nine naive MCP-1−/− mice were challenged by injecting NP-O-Su in one footpad. 24 h later, the difference in thickness between injected and noninjected footpads was determined. Annotations are the same as in A. Similar results were observed in males and females. (C) F4/80+ cells in contact hypersensitivity lesions. Contact hypersensitivity challenges were performed as described in A, and ears from sensitized mice were harvested 24 h after challenge. Sections were stained and the proportion of F4/80+ cells from two animals in each group were determined. Error bars indicate SEM. These results are typical of two independent experiments.
Figure 4
Figure 4
Secondary granuloma formation in response to Schistosoma mansoni eggs in wild-type and MCP-1−/− mice. Synchronous pulmonary granulomata were induced in chronically infected wild-type (+/+) or MCP-1−/− (−/−) mice as described in Materials and Methods. At the indicated time after egg injection, lungs from five to six mice were collected and the granuloma area was determined. Differences between wild-type and MCP-1−/− mice were significant at day 2 (P <0.01) and day 8 (P <0.001) by Student's t test. Error bars indicate SEM. At least 20 granulomata were measured per mouse.
Figure 5
Figure 5
CFU in organs of wild-type and MCP-1–deficient mice inoculated with Mycobacterium tuberculosis. 16 wild-type and 16 MCP-1−/− mice were injected intravenously with 105 CFU of Mycobacterium tuberculosis. At the indicated days after infection, 4 mice from each group were sacrificed and homogenates of liver (A), spleen (B), and lung (C) were plated. CFU are indicated from wild type (black square) and MCP-1−/− (gray circle) mice. Error bars indicate SEM. *P ⩽0.05, **P ⩽0.025 by Student's t test.
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