Role of the SDF-1/CXCR4 axis in the pathogenesis of lung injury and fibrosis

Abstract

Stromal cell-derived factor-1 (SDF-1) participates in mobilizing bone marrow-derived stem cells, via its receptor CXCR4. We studied the role of the SDF-1/CXCR4 axis in a rodent model of bleomycin-induced lung injury in C57BL/6 wild-type and matrix metalloproteinase (MMP)-9 knockout mice. After intratracheal instillation of bleomycin, SDF-1 levels in serum and bronchial alveolar lavage fluid increased. These changes were accompanied by increased numbers of CXCR4(+) cells in the lung and a decrease in a population of CXCR4(+) cells in the bone marrow that did not occur in MMP-9(-)/(-) mice. Both SDF-1 and lung lysates from bleomycin-treated mice induced migration of bone marrow-derived stem cells in vitro that was blocked by a CXCR4 antagonist, TN14003. Treatment of mice with TN14003 with bleomycin-induced lung injury significantly attenuated lung fibrosis. Lung tissue from patients with idiopathic pulmonary fibrosis had higher numbers of cells expressing both SDF-1 and CXCR4 than did normal lungs. Our data suggest that the SDF-1/CXCR4 axis is important in the complex sequence of events triggered by bleomycin exposure that eventuates in lung repair. SDF-1 participates in mobilizing bone marrow-derived stem cells, via its receptor CXCR4.

Figures

Figure 1.

SDF-1 expression in lungs of mice after bleomycin treatment. After 0, 1, 3, 7, and 14 d of bleomycin treatment, mice were killed. Serum (A) and BAL (B) SDF-1 levels were determined by ELISA. Values represent mean ± SE (n = 6, *P < 0.05).

Figure 2.

CXCR-4 expression in mice after bleomycin treatment. Female C57BL/6 mice, 8 wk old, were given 4 U/kg bleomycin in 100 μl of PBS or 100 μl of PBS alone intratracheally. After 0, 3, 7, and 14 d, mice were killed. (A) CXCR4 protein expression as shown in Western blot. (B) Quantitation of CXCR4 levels from Western blot by densitometry. Basal level was designated as 1. Values represent mean ± SE (n = 4, *P < 0.05).

Figure 3.

FACS analysis of BM cells. (A) Total bone marrow cells were labeled with anti-mouse CXCR4. Both labeled (blue curve) and unlabeled (red curve) samples were subjected to FACS analysis. (B) Total bone marrow cells were labeled with anti-mouse CD45, CD11b, and CXCR4. Samples were subjected to FACS analysis. (C) CD45− fraction of BM cells was purified by MACS separation system. The resulting cells were labeled with anti-mouse CD45, CD105, CD44, and CXCR4 antibodies and gated for CD45− cells. Data shown in left panel were dual expression of CD105 and CD44. In the right panel, the CD45−CD105+CD44+ fractions were further examined for the expression of CXCR4 (solid line). Flow cytometry data were generated using the FlowJo 6.1.1 software.

Figure 4.

Effect of MMP-9 on recruitment of CD45−CD105+CD44+CXCR4+ cells from BM after bleomycin treatment. After 0, 3, and 7 d of bleomycin treatment, BM samples were harvested and CD45− fraction collected by MACS separation. The samples were stained with anti-mouse CD45, CD105, CD44, and CXCR4 antibodies and analyzed by FACS. (A) In wild-type C57BL/6 mice, FACS data showed changes of CD45−CD105+CD44+expression at different time points. (B) Results from MMP-9−/− C57BL/6 mice. (C) MMP-9 activity in BM stromal supernatant as determined by gelatin zymography. Clear bands revealed MMP gelatinase activity.

Figure 5.

Effect of lung lysate on MSC migration. Cultured bone marrow cells were depleted with CD45 and CD11b antibodies to purify MSC (1 × 105) and plated in Matrigel-coated transwells. Minimal essential medium was added to both upper and lower chambers. Extracts of lungs harvested from mice 3 d after bleomycin or PBS treatment (200 μg of total protein), SDF-1 (200 ng/ml), MIP-2 (100 ng/ml) or PBS was added to the lower chamber. For effects of the CXCR4 antagonist on stem cell migration, cells were incubated with 1 μM of TN14003 for 30 min at 37°C before migration. Migration assays were performed at 37°C for 2 h. Transmigrated cells were collected and quantified by a hemocytometer. Values represent mean ± SE (*,**P < 0.01, n = 4).

Figure 6.

A CXCR4 antagonist inhibits bleomycin induced pulmonary fibrosis. Mice were treated with bleomycin in the presence of CXCR4 antagonist, TN14003 or PBS. (A) Frozen lung sections from mice treated with bleomycin on Days 0 and 20 were stained with a rabbit anti-CXCR4 antibody or a control rabbit IgG (control). (B) Representative H&E-stained histopathologic sections of saline-treated (left panels) or TN14003-treated (right panels) lung tissues on Day 20 after bleomycin treatment. (C) Masson's Trichrome staining of lung sections from the same experimental groups. (D) Collagen content of lung tissue as determined by hydroxyproline assay. *= significant increase as compared to control, ** = significant reduction as compared to bleomycin treatment. Values represent mean ± SE (*,**P < 0.05, n = 5).

Figure 7.

Increased SDF-1 and CXCR-4 in IPF lung samples compared with normal human lungs. Lung tissues from patients with IPF (right panels) and from normal control subjects (left panels) were paraffin embedded and sectioned. Immunohistochemistry was performed for SDF-1 and CXCR-4. Cells stained dark brown were SDF-1– (inside and around a vessel) or CXCX4 (interstitium)-expressing cells (see arrows).