Inhibitory effect of CXC chemokine receptor 4 antagonist AMD3100 on bleomycin induced murine pulmonary fibrosis

Abstract

CXC chemokine receptor 4 (CXCR4), which binds the stromal cell-derived factor-1 (SDF-1), has been shown to play a critical role in mobilizing the bone marrow (BM)-derived stem cells and inflammatory cells. We studied the effects of AMD3100, CXCR4 antagonist, on a murine bleomycin-induced pulmonary fibrosis model. Treatment of mice with AMD3100 in bleomycin-treated mice resulted in the decrease of SDF-1 in bronchoalveolar lavage (BAL) fluids at an early stage and was followed by the decrease of fibrocytes in the lung. AMD3100 treatment decreased the SDF-1 mRNA expression, fibrocyte numbers in the lung at an early stage (day 3) and CXCR4 expression at the later stage (day 7 and 21) after bleomycin injury. The collagen content and pulmonary fibrosis were significantly attenuated by AMD3100 treatment in later stage of bleomycin injury. AMD3100 treatment also decreased the murine mesenchymal and hematopoietic stem cell chemotaxis when either in the stimulation with bleomycin treated lung lysates or SDF-1 in vitro. In BM stem cell experiments, the phosphorylation of p38 MAPK which was induced by SDF-1 was significantly blocked by addition of AMD3100. Our data suggest that AMD3100 might be effective in preventing the pulmonary fibrosis by inhibiting the fibrocyte mobilization to the injured lung via blocking the SDF-1/CXCR4 axis.

Figure 1

The total inflammatory cell and differential cell numbers in the BAL fluids were measured from the mice. Total inflammatory cells were increased by AMD3100 treatment at day 3 and 21 than bleomycin injury alone (^*: P < 0.05). No difference of neutrophil counts ware noted between bleomycin group and bleomycin plus AMD3100 group.

Figure 2

The SDF-1 (A), TGF-β1 (B) and KC (C) concentrations were significantly higher in the BAL fluids of bleomycin-injury group than in the control mice. AMD3100 treatment decreased the SDF-1 (A) and KC (C) concentrations on day 3 after bleomycin injury but had no effects on the TGF-β1 (B) concentrations. Data points and error bars correspond to the means + SE. n = 6 animals.group^-1 (^*: P < 0.05; ^**: P < 0.01).

Figure 3

Intrapulmonary CD45+CXCR4+Col I+ fibrocytes recruitment after bleomycin injury. CD45+CXCR4+Col I+ fibrocytes were significantly increased on day 3 after bleomycin injury. AMD3100 treatment significantly decreased the fibrocytes recruitment on day 3 after bleomycin injury (A, B). Single cell suspensions from the lung in the bleomycin injury group, bleomycin plus AMD3100 group and control group were made and triple stained for CD45, Col I, and CXCR4, then examined by FACS analysis. n = 5 samples.group^-1 (^*: P < 0.05; ^**: P < 0.01).

Figure 4

Collagen content and representative histopathology. The collagen content in the lung was increased from 3 days after bleomycin injury until day 21. The collagen content was decreased by AMD3100 treatment on day 21 after bleomycin injury (A). Total collagen content was determined by the Sircol assay. Severe pulmonary inflammation and fibrosis occurred after bleomycin injury on day 21 and this histological change was markedly reduced by AMD3100 treatment (B). Pulmonary fibrosis was scored by Ashcroft method. Representative photomicrographs of lung tissue stained with a hematoxyline-eosin (C). Original magnification X 200. n = 4 lungs.group^-1 (^*: P < 0.05; ^**: P < 0.01).

Figure 5

Expression of SDF-1 mRNA in mouse lung. SDF-1 mRNA expression in the lung was increased at 0, 3 and 7 days after bleomycin injury than in control mice. AMD3100 treatment decreased the SDF-1 mRNA expression in the lung at 0 and 3 days after bleomycin injury. In each group, mRNA levels were analyzed using real-time RT-PCR and β-actin as housekeeping gene. Nonparametric Kruskal-Wallis H test. n = 3 lungs in each group (^*: P < 0.05).

Figure 6

Western immunoblotting showing CXCR4 protein in the lung was increased from 3 days to 21 days after bleomycin injury. AMD3100 treatment decreased the CXCR4 protein at 7 and 21 days after bleomycin injury. C: control, B: bleomycin, BA: bleomycin plus AMD3100. Nonparametric Kruskal-Wallis H test. n = 3 lungs.group^-1 (^*: P < 0.05).

Figure 7

Chemotaxis of MSCs and HSCs across the filter of HTS Transwell®-96 Permeable Support Systems. The MSC and HSC were collected from the mouse femur and allowed to migrate to the lower chamber toward several stimulants in Transwell Systems. Cells that migrated to the lower chamber were counted microscopically. SDF-1 (50 ng/ml) and bleomycin-injured lung lysate markedly increased the migration of MSCs (A) and HSCs (B). Adding the AMD3100 (100 µg/ml) to the bleomycin-injured lung lysates or SDF-1 treated samples inhibited the stem cell migration. Lung lysates from the AMD3100 treated groups in bleomycin-injured mice also showed inhibition of stem cell migration compared with bleomycin-injured lung lysates. Nonparametric Kruskal-Wallis H test. Results are mean of at least three independent experiments (^*: P < 0.05; ^**: P < 0.01).

Figure 8

MAPK phosphorylation analysis of mesenchymal stem cells. The SDF-1 phosphorylated the p38 MAPK in MSCs (A) and HSCs (B). The phosphorylation level of p38 MAPK was apparently decreased by addition of AMD3100 as much as SB203580, a specific p38 MAPK inhibitor, in mice MSCs and HSCs. The mesenchymal or hematopoitic stem cells were purified from the mouse femur. Results are representative of at least three independent experiments (^*: P < 0.05; ^**: P < 0.01).