Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis - PubMed (original) (raw)

Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis

Roderick J Phillips et al. J Clin Invest. 2004 Aug.

Abstract

Previous reports have identified a circulating pool of CD45(+) collagen I(+) CXCR4(+) (CD45(+)Col I(+)CXCR4(+)) cells, termed fibrocytes, that traffic to areas of fibrosis. No studies have demonstrated that these cells actually contribute to fibrosis, however. Pulmonary fibrosis was originally thought to be mediated solely by resident lung fibroblasts. Here we show that a population of human CD45(+)Col I(+)CXCR4(+) circulating fibrocytes migrates in response to CXCL12 and traffics to the lungs in a murine model of bleomycin-induced pulmonary fibrosis. Next, we demonstrated that murine CD45(+)Col I(+)CXCR4(+) fibrocytes also traffic to the lungs in response to a bleomycin challenge. Maximal intrapulmonary recruitment of CD45(+)Col I(+)CXCR4(+) fibrocytes directly correlated with increased collagen deposition in the lungs. Treatment of bleomycin-exposed animals with specific neutralizing anti-CXCL12 Ab's inhibited intrapulmonary recruitment of CD45(+)Col I(+)CXCR4(+) circulating fibrocytes and attenuated lung fibrosis. Thus, our results demonstrate, we believe for the first time, that circulating fibrocytes contribute to the pathogenesis of pulmonary fibrosis.

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Figures

Figure 1

Figure 1

Characterization of human fibrocytes and their trafficking to bleomycin-induced lung fibrosis in SCID mice. (A) Human fibrocytes stained with control Ab, human Col I, human CXCR4, and human α-SMA 3 weeks after purification. Representative fields viewed at ×400 magnification. (B) Isolated human fibrocytes triple-stained for Col I, CD45, and CXCR4 were examined by FACS analysis. Data shows CD45+ fibrocytes that were examined for dual expression of CXCR4 and Col I. (C) Isolated human fibrocyte chemotaxis in response to 0, 30, and 100 ng/ml of CXCL12. Data are representative of three experiments. *P < 0.05. h.p.f., high-powered field. (D) Isolated human fibrocytes (106) were injected into the tail vein of SCID mice at day 4 after treatment with either intratracheal bleomycin (Bleo) or saline, followed by recovery of cells from the lungs at day 8. Cells were then stained for human CD45, Col I, and CXCR4 and analyzed by flow cytometry to determine trafficking of human fibrocytes to the lungs of SCID mice. *P < 0.05.

Figure 2

Figure 2

Kinetics of Col I and Col III gene expression and total collagen protein deposition in the lung during bleomycin-induced pulmonary fibrosis. (A and B) Kinetics of pro-Col I (A) and pro–Col III (B) gene expression in lungs of mice exposed to intratracheal bleomycin compared with saline control as determined by real-time quantitative PCR. n = 6 lungs in each group. Data represent the mean ± SEM. *P < 0.05, significant differences between bleomycin and saline groups. ct, threshold cycle number. (C) Kinetics of total collagen protein deposition in lungs of mice exposed to either intratracheal bleomycin, saline, or naive (time 0) control as determined by the Sircol assay. n = 6 lungs in each group. Data represent the mean ± SEM. *P < 0.05, significant differences between bleomycin and saline groups.

Figure 3

Figure 3

Intrapulmonary recruitment of CD45+Col I+CXCR4+ fibrocytes is greater than CD45+Col I+CCR7+ fibrocytes and correlates with collagen deposition in the lungs of bleomycin-exposed mice. (A and B) Single-cell suspensions were isolated from bleomycin- or saline-challenged lungs and blood buffy coats at the times indicated, triple-stained for CD45, Col I, and CXCR4 (A), or CD45, Col I, and CCR7 (lungs only) (B), and then examined by FACS analysis. n = 6 samples per group. BC, buffy coat. Data represent the mean ± SEM. *P < 0.05, significant differences between bleomycin and saline groups. **P < 0.05, significant differences between saline-exposed mice and the naive mice. (C) Mice were treated with either intratracheal bleomycin or saline for 8 days. Bone marrow was removed, triple-stained for CD45, Col I, and CXCR4, and then examined by FACS analysis. n = 3 samples per group. Data represent the mean ± SEM. *P < 0.05. (D) Kinetics of CXCL12 protein expression in lung tissue and plasma of mice exposed to either intratracheal bleomycin, saline, or naive control (day 0) as determined by ELISA. n = 6 samples in each group. Data represents the mean ± SEM. *P < 0.05, significant differences between bleomycin and saline groups. **P < 0.05, significant differences between saline-exposed mice and the naive mice.

Figure 4

Figure 4

Neutralizing anti-CXCL12 Ab’s inhibit intrapulmonary recruitment of CD45+Col I+CXCR4+ fibrocytes and significantly attenuate lung fibrosis in bleomycin-treated mice. (A) Single-cell suspensions were isolated from the lungs of naive or saline- or bleomycin-exposed mice, where the bleomycin-treated mice also received daily injections of either anti-CXCL12 Ab or control Ab for 16 days. These cells were then triple-stained for CD45, Col I, and CXCR4 and analyzed by flow cytometry. n = 5 samples per group. Data represent the mean ± SEM. *P < 0.05. (B) Total collagen present in lung tissue of naive, saline-, or bleomycin-exposed mice, where the bleomycin-treated mice also received daily injections of either anti-CXCL12 or control Ab’s for 16 days. Total collagen levels were determined by the Sircol assay. n = 4 lungs in each group. Data represent the mean ± SEM. *P < 0.05. (C) Single-cell suspensions were isolated from the lungs of bleomycin-exposed mice treated with either daily injections of anti-CXCL12 or control Ab’s for 8 days. These cells were then individually stained for CD3, CD4, CD8, NK1.1, Ly6, and Mac519 (Mac) and analyzed by flow cytometry. n = 3 lungs per group.

Figure 5

Figure 5

Representative histopathology and morphometric analysis of picrosirius red in bleomycin-induced pulmonary fibrosis and expression of α-SMA in the presence of neutralizing anti-CXCL12 or control Ab’s. (A) Representative H&E-stained histopathologic sections of lung tissue on day 16 after intratracheal bleomycin administration in the presence of daily injections of either control (upper panel) or neutralizing anti-CXCL12 Ab’s (lower panel). (B) Morphometric analysis of picrosirius red–stained lung tissue was measured by image analysis (NIH Image 1.55) and expressed as area (square pixels) at ×400 magnification. *P < 0.001. (C) Histopathologic sections of lung tissue on day 16 after intratracheal bleomycin administration in the presence of daily injections of either control (left panel) or neutralizing anti-CXCL12 Ab’s (right panel) stained with α-SMA. Representative fields viewed at ×400 magnification.

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