A novel mechanism for CCR4 in the regulation of macrophage activation in bleomycin-induced pulmonary fibrosis

Abstract

Macrophage polarization into M1 or M2 phenotypes dictates the nature, duration, and severity of an inflammatory response. The objective of this study was to examine the role of CC chemokine receptor 4 (CCR4) in macrophage polarization during pulmonary oxidative injury in wild-type [WT (CCR4(+/+))] and CCR4-deficient (CCR4(-/-)) mice. Intrapulmonary administration of bleomycin sulfate provoked lethal inflammatory and fibrotic responses in WT (CCR4(+/+)) mice, but such responses were absent in CCR4(-/-) mice. Transcript and protein analyses of alveolar and bone marrow-derived macrophages showed that cells isolated from CCR4(-/-) mice did not exhibit CCL17-dependent M1 activation in response to bleomycin. Instead, CCR4(-/-) macrophages showed an M2 phenotype characterized by significantly elevated expression of arginase 1 and FIZZ1 (found in inflammatory zone 1), particularly during the peak of pulmonary inflammation. Compared with WT (CCR4(+/+)) mice, CCR4(-/-) mice exhibited a significant increase in the expression of the nonsignaling CC chemokine scavenging receptor D6 in whole lung samples and isolated macrophages. Thus, these results demonstrate that CCL17-dependent activation of CCR4 in macrophages plays a central role in free radical-induced pulmonary injury and repair.

Figure 1

CCR4^−/− mice were protected from the lethal and pulmonary remodeling effects of bleomycin challenge. A: Percent survival of WT versus CCR4^−/− mice after bleomycin challenge. B: Histological analysis of lungs and quantification of collagen deposition after bleomycin challenge. Representative H&E-stained histological sections: untreated WT (CCR4^+/+) mouse lung (a), untreated CCR4^−/− mouse lung (c), WT (CCR4^+/+) mouse lung at day 21 after bleomycin (e), CCR4^−/− mouse lung at day 21 after bleomycin (g). Representative Masson-trichrome histological sections: untreated WT (CCR4^+/+) (b), untreated CCR4^−/− mouse lung (d), WT (CCR4^+/+) mouse lung at day 21 after bleomycin (f), CCR4^−/− mouse lung at day 21 after bleomycin (h). C: Hydroxyproline levels in whole lung homogenates from untreated or bleomycin-challenged mice. Data are mean ± SEM from four to five mice at each time point. ***P < 0.001, **P < 0.01.

Figure 2

Quantitative TaqMan PCR and ELISA analysis of CCL17 and CCL22 expression in whole lung samples after bleomycin challenge. CCL17 transcript expression in whole lung homogenates at days 1, 3, 7, or 21 after bleomycin (A), and in bone marrow-derived macrophages at the same times after bleomycin (B). CCL22 transcript expression in whole lung homogenates at days 1, 3, 7, or 21 after bleomycin (C), and in bone marrow-derived macrophages at the same times after bleomycin (D). For all TaqMan analyses, the data were expressed as the fold-increase in transcript expression above transcript levels measured in whole lung or macrophage samples before bleomycin challenge. ELISA analysis of CCL17 (E) and CCL22 (F) in whole lung homogenates from WT (CCR4^+/+) and CCR4^−/− mice at day 21 after the bleomycin challenge. Data are mean ± SEM from four to five mice at each time point. **P < 0.01. N.S. = not significant.

Figure 3

Cell quantification in BAL samples at days 1, 3, and 7 after bleomycin challenge. Flow cytometric analyses: relative numbers of DCs, monocytes/macrophages, granulocytes, and alveolar macrophages in the BAL at day 1 after bleomycin challenge based on CD11b and CD11c expression (A), and the presence (B, macrophages) and absence of MHC II (C, granulocytes). D: Cytospin analysis. BAL samples were collected at days 1, 3, and 7 after bleomycin challenge, centrifuged onto glass slides, stained with Diff-Quik, and then visualized by light microscopy at ×200 magnification. The representative photomicrographs shown are as follows: a: WT (CCR4^+/+) day 1; b: CCR4^−/− day 1; c: WT (CCR4^+/+) day 3; d: CCR4^−/− day 3; e: WT (CCR4^+/+) day 7; and f: CCR4^−/− day 7.

Figure 4

Determination of the BAL macrophage phenotype in WT (CCR4^+/+) and CCR4^−/− during the acute inflammatory phase. Cells present in the BAL from WT (CCR4^+/+) and CCR4^−/− mice before and at days 1, 3, and 7 after bleomycin challenge were plated in tissue culture plates at a density of 2.5 × 10⁵ cells/ml in RPMI plus 15% fetal calf serum and incubated for 1 hour at 37°C. Nonadherent cells were discarded and adherent cells were subjected to RNA isolation for quantitative TaqMan PCR analysis of Arg1 (A), FIZZ1 (B), and NOS2 (C) transcript expression. Data are mean ± SEM from BAL macrophages cultured from five mice at each time point. ***P < 0.001. N.S. = not significant.

Figure 5

Effect of CCL17 and CCL22 stimulation on NOS2 and Arg1 transcript expression in bone marrow-derived macrophages. Bone marrow-derived macrophages from WT (CCR4^+/+) and CCR4^−/− mice at day 1 after bleomycin challenge were stimulated with one of the following: media (RPMI + 0.5% fetal calf serum), 10 ng/ml of CCL22, or 10 ng/ml CCL17 for 24 hours at 37°C. NOS2 (A) and Arg1 (B) transcript expression was measured using quantitative TaqMan PCR analysis. For all TaqMan analyses, the data were expressed as the fold-increase in transcript expression above transcript levels measured in untreated macrophage samples at day 1 after bleomycin challenge. Data are mean ± SEM from bone marrow-derived macrophages cultured from five mice at each time point. **P < 0.01. N.S. = not significant.

Figure 6

Immunoreactive CC chemokine ligand levels in whole lung homogenates from WT (CCR4^+/+) and CCR4^−/− mice before and at days 1, 3, and 7 days after bleomycin challenge. Significantly elevated levels of CCL2 (A), CCL3 (B), CCL17 (C), and CCL22 (D) were measured in whole lung homogenates from WT (CCR4^+/+) mice at day 1 after bleomycin challenge compared to levels of these chemokines in whole lung samples from CCR4^−/− mice before the bleomycin challenge. Data are mean ± SEM from four to five mice at each time point. ***P < 0.001, **P < 0.01, *P < 0.05. N.S. = not significant.

Figure 7

D6 transcript expression by BAL macrophages and bone marrow macrophages before and at day 1 after bleomycin challenge. A: FITC-CCL17 binding to WT (CCR4^+/+) and CCR4^−/− BAL macrophages. B: D6 transcript expression in BAL WT and CCR4^−/− macrophages before and at day 1 after bleomycin was measured using TaqMan PCR analysis. C: FITC-CCL17 binding to WT (CCR4^+/+) in the presence of control IgG or a blocking antibody against CCR4. D: FITC-CCL17 binding to CCR4^−/− BAL macrophages in the presence of control IgG or a blocking antibody against D6. BAL macrophages before (E) and at day 1 (F) after bleomycin were cultured with media alone, or with media containing 10 ng/ml of CCL22 or 10 ng/ml of CCL17 for 24 hours and D6 transcript expression was measured. G: Bone marrow macrophages isolated day 1 after bleomycin were cultured and treated with media alone, or with media containing 10 ng/ml of CCL22 or 10 ng/ml of CCL17 for 24 hours and D6 transcript expression was measured. D6 transcript expression was measured using TaqMan and data were expressed as the fold-increase in transcript expression above transcript levels measured in untreated macrophage samples before or at day 1 after bleomycin. Data are mean ± SEM from bone marrow-derived macrophages cultured from five mice at each time point. ***P < 0.001, **P < 0.01, *P < 0.1.

Figure 8

Immunolocalization and quantitative TaqMan analysis of D6 in bleomycin-treated lung. A: Representative histological lung sections from WT (CCR4^+/+) and CCR4^−/− mice at days 1 [WT (CCR4^+/+) (b), CCR4^−/− (e)] and 21 [WT (CCR4^+/+) (c), CCR4^−/− (f)] after bleomycin administration were stained with a goat anti-mouse D6 antibody and processed using routine immunohistochemistry techniques. Control antibody staining in representative whole lung samples WT (CCR4^+/+) (a) and CCR4^−/− (d) mice at day 1 after bleomycin. B: D6 transcript expression in whole lungs from WT (CCR4^+/+) and CCR4^−/− before and at day 21 after bleomycin challenge was measured using quantitative TaqMan PCR analysis. The data were expressed as the fold-increase in transcript expression above transcript levels measured in untreated macrophage samples before bleomycin challenge. Data are mean ± SEM from bone marrow-derived macrophages cultured from five mice at each time point. **P < 0.01.

Figure 9

Proposed model for the role of CCR4 in the activation of macrophages in bleomycin-induced pulmonary fibrosis. CCR4^+/+: In the presence of CCR4, CCL17 and CCL22 promote tissue injury through the induction of the M1 macrophage phenotype (increased NOS2 and decreased Arg1 expression). CCR4^−/−: In the absence of CCR4, the M1 macrophage activation occurs through the suppression of NOS2 and increased Arg1 and FIZZ1 expression. The M2 macrophage phenotype favors the up-regulation of D6, which attenuates inflammation and tissue injury.