Fibrocytes are a potential source of lung fibroblasts in idiopathic pulmonary fibrosis

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Abstract

Idiopathic pulmonary fibrosis is characterized by the accumulation of fibroblasts/myofibroblasts and aberrant remodeling of the lung parenchyma. However, the sources of fibroblasts in IPF lungs are unclear. Fibrocytes are circulating progenitors of fibroblasts implicated in wound healing and fibrosis. In this study we evaluated evidence for the presence of fibrocytes in the lung of patients with idiopathic pulmonary fibrosis by immunofluorescence and confocal microscopy. Fibrocytes were identified in tissues in 8 out of 9 fibrotic lungs. Combinations including CXCR4 and a mesenchymal marker stained significantly more fibrocytes/mm2 of tissue compared with combinations using CD34 or CD45RO with mesenchymal markers: CXCR4/procollagen-I (10.3 ± 2.9 fibrocytes/mm2) and CXCR4/prolyl-4-hydroxylase (4.1 ± 3.1), versus CD34/procollagen-I (2.8 ± 3.0), CD34/αSMA (2.2 ± 1.6) and CD45RO/prolyl-4-hydroxylase (1.3 ± 1.6); p < 0.003. There was a positive correlation between the abundance of fibroblastic foci and the amount of lung fibrocytes (r = 0.79; p < 0.02). No fibrocytes were identified in normal lungs. The fibrocyte attractant chemokine CXCL12 increased in plasma [median: 2707.5 pg/ml (648.1–4884.7) versus 1751.5 pg/ml (192.9–2686.0) from healthy controls; p < 0.003)] and was detectable in the bronchoalveolar lavage fluid of 40% of the patients but not in controls. In the lung CXCL12 was strongly expressed by alveolar epithelial cells. A negative correlation between plasma levels of CXCL12 with lung diffusing capacity for carbon monoxide (DLCO) (r = −0.56; p < 0.03) and oxygen saturation on exercise was found (r = −0.41; p < 0.04). These findings indicate that circulating fibrocytes, likely recruited through the CXCR4/CXCL12 axis, may contribute to the expansion of the fibroblast/myofibroblast population in idiopathic pulmonary fibrosis.

Introduction

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually lethal lung disease of unknown etiology (Gross & Hunninghake, 2001). Expansion of the fibroblast/myofibroblast population with the formation of fibroblastic foci in the lung is a characteristic of the disease (Gross & Hunninghake, 2001; Selman, King, & Pardo, 2001; Thannickal, Toews, White, Lynch, & Martinez, 2004).

In the past few years, the origin of lung fibroblasts in IPF has been debated. While the prevailing view was that in response to injury resident mesenchymal cells migrated, proliferated and synthesized extracellular matrix, it has recently been suggested that mesenchymal cells in IPF lungs may be derived from epithelial to mesenchymal transition (Kim et al., 2006, Willis et al., 2005) as well as bone marrow-derived mesenchymal cells (Epperly, Guo, Gretton, & Greenberger, 2003; Hashimoto, Jin, Liu, & Chensue, 2004).

Another potential extra-pulmonary source of fibroblasts/myofibroblasts is the fibrocyte, a distinct population of circulating mesenchymal progenitors that display a unique cell surface phenotype (Quan, Cowper, Wu, Bockenstedt, & Bucala, 2004). They co-express the common leukocyte antigen CD45, the hematopoietic stem cell antigen CD34, and mesenchymal markers such as collagen I, fibronectin, α-smooth muscle actin (α-SMA) and prolyl-4-hydroxylase (Aiba & Tagami, 1997; Quan et al., 2004; Strieter, Gomperts, & Keane, 2007). Circulating fibrocytes rapidly enter sites of tissue injury, and may contribute to scar formation (Postlethwaite, Shigemitsu, & Kanangat, 2004). Patients with asthma show accumulation of fibrocytes in the bronchial mucosa that localize to areas of fibrosis below the epithelium (Nihlberg et al., 2006; Schmidt, Sun, Stacey, Mori, & Mattoli, 2003). Also, infiltrating fibrocytes have been demonstrated in fibrotic lung tissue of mice injured by fluorescein isothiocyanate or bleomycin (Moore et al., 2006, Phillips et al., 2004). Recently, it was reported that patients with IPF and fibrotic non-specific interstitial pneumonia have increased numbers of circulating fibrocytes (Mehrad et al., 2007). Importantly, most of them expressed the receptor CXCR4 supporting the notion that the CXCR4/CXCL12 pathway may be important for tissue recruitment (Mehrad et al., 2007, Moore et al., 2006, Strieter et al., 2007). So far, fibrocytes have not been demonstrated in the lungs of patients with IPF or other human interstitial lung diseases.

We hypothesized that fibrocytes are present and contribute to the fibroblast/myofibroblast expansion in the IPF lungs. To that end, we evaluated the presence of these cells in IPF and control lungs by immunofluorescence and confocal microscopy. We also quantified the plasma and bronchoalveolar lavage levels, and examined the lung localization of stromal-derived factor-1 (SDF-1/CXCL12), a specific ligand for CXCR4.

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Materials and methods

Forty-two untreated IPF patients (27 males, 15 females; 65.7 ± 10.3 years old) were included in this study. Diagnosis of IPF was based on established criteria and confirmed by open lung biopsy showing microscopic findings consistent with usual interstitial pneumonia (American Thoracic Society (ATS) and European Respiratory Society (ERS), 2000). Clinical data, pulmonary function tests and bronchoalveolar lavage (BAL) cell profile data were extracted from case records. The research protocol was

Fibrocytes are revealed in IPF lungs but are absent in BAL

The presence of fibrocytes was determined in 9 IPF and 4 control lungs by confocal microscopy using five different specific combinations for fibrocytes: CD34/procollagen-I, CD34/αSMA, CD45RO/prolyl-4-hydroxylase, CXCR4/procollagen-I, and CXCR4/prolyl-4-hydroxylase. Fibrocytes were found in varying numbers in 8 of 9 untreated IPF patients (Table 2). The combination of CXCR4 and any mesenchymal marker identified significantly more fibrocytes/mm2 of tissue as compared to marker combinations of

Discussion

IPF is characterized by the formation of fibroblastic/myofibroblastic foci and by a progressive fibrotic remodeling which result in the irreversible destruction of the lung architecture. However, the source of fibroblasts/myofibroblasts in this disease remains unresolved. It is well known that fibroblasts obtained from IPF lungs are phenotypically and functionally heterogeneous (Phan, 2003). This heterogeneity may reflect not only a diverse activation and differentiation processes that take

Conflict of interest

The authors declare that no financial or other potential conflicts of interest exist.

Acknowledgements

This study was supported by Universidad Nacional Autónoma de México, Grant SDI.PTID.05.6, by the Swedish Medical Research Council (11550), Heart-Lung Foundation, CFN—Central Försöksdjursnämnden, Alfred Österlund Foundation and Greta and Johan Kock Foundation, Medical Faculty of Lund University, Sweden, and by a Grant for research from Pfizer Institute, Mexico. The authors thank Saana Karttunen and Professor Juha Risteli, Oulu University, Finland, for generously providing the antibodies directed

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    These authors contributed equally to this study.

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