Role of chemokine ligand 2 in the protective response to early murine pulmonary tuberculosis

Abstract

Chemokines play an important role in the development of immunity to tuberculosis. Chemokine ligand 2 (CCL2, JE, monocyte chemoattractant protein-1) is thought to be primarily responsible for recruiting monocytes, dendritic cells, natural killer cells and activated T cells, all of which play critical roles in the effective control of tuberculosis infection in mice. We show here that in mice in which the CCL2 gene was disrupted, low-dose aerosol infection with Mycobacterium tuberculosis resulted in fewer macrophages entering the lungs, but only a minor and transient increase in bacterial load in the lungs; these mice were still able to establish a state of chronic disease. Such animals showed similar numbers of activated T cells as wild-type mice, as determined by their expression of the CD44hi CD62lo phenotype, but a transient reduction in cells secreting interferon-gamma. These data indicate that the primary deficiency in mice unable to produce CCL2 is a transient failure to focus antigen-specific T lymphocytes into the infected lung, whereas other elements of the acquired host response are compensated for by different ligands interacting with the chemokine receptor CCR2.

Figure 1

Mice lacking the gene for chemokine ligand 2 (CCL2) differ in their susceptibility to low-dose aerosol infection with Mycobacterium tuberculosis. Control (black bars) and CCL2 gene knockout (CCL2-KO) (white bars) mice were infected via the aerogenic route. The lungs (a), spleen (b) and liver (c) were evaluated for bacterial growth at different time points after infection. Data points represent the mean (± standard error of the mean) values from four mice. The value marked with an asterisk is significantly different from that of control mice at the same time point (*P = 0·019, Students's t-test). Data are representative of three separate experiments.

Figure 2

Mice lacking the gene for chemokine ligand 2 (CCL2) were able to activate T cells similar to control mice following aerogenic challenge with Mycobacterium tuberculosis. Control (black bars) or CCL2-knockout (white bars) mice were infected as described in the legend to Fig. 1, and splenocytes were analysed by flow cytometry for expression of CD4⁺ CD44^hi CD62L^lo (a) and CD8⁺ CD44^hi CD62L^lo (b). The number of cells expressing high levels of CD44 and low levels of CD62L per spleen is reported. Splenocytes were also stimulated with anti-CD3, anti-CD28 and monensin, and then analysed for intracellular interferon-γ (IFN-γ) production (c and d). The bars represent the mean values of four mice per group, and the graph shows one experiment representative of three.

Figure 3

Mice lacking the gene for chemokine ligand 2 (CCL2-KO) fail to recruit as many interferon-γ (IFN-γ)-producing T lymphocytes to the lung as wild-type mice. Control (black bars) or CCL2-KO (white bars) mice were infected as described in the legend to Fig. 1, and lung cells were analysed by flow cytometry for expression of CD4⁺ CD44^hi CD62L^lo (a) and CD8⁺ CD44^hi CD62L^lo (b). The number of cells expressing high levels of CD44 and low levels of CD62L per lung is reported. Lung cells were also stimulated with anti-CD3, anti-CD28 and monensin, and then analysed for intracellular IFN-γ production (c and d). The bars represent the mean value of four mice per group, and the graph shows one experiment representative of three. The presence of an asterisk indicates statistical significance between control and CCL2-KO mice (*P < 0·05, Student's t-test).

Figure 4

Representative micrograph showing lung lesions in C57BL/6 mice (a and c) and chemokine ligand 2 knockout (CCL2-KO) mice (b and d) 70 days after aerosol infection with Mycobacterium tuberculosis (haematoxylin and eosin staining). Size bars are 1-mm (a and b), 100-µm (c and d), and 10-µm insets. Note the macrophage-rich granulomatous response (asterisk and inset of c) in C57BL/6 mice, while in CCL2-deficient mice there is a predominant accumulation of lymphocytes (arrows and inset of d).