Abstract and Introduction
Abstract
Background Bone marrow-derived fibrocytes reportedly play important roles in the pathogenesis of idiopathic pulmonary fibrosis. Pirfenidone is an anti-fibrotic agent; however, its effects on fibrocytes have not been investigated. The aim of this study was to investigate whether pirfenidone inhibits fibrocyte pool size in the lungs of bleomycin-treated mice.
Methods Bleomycin (100 mg/kg) was infused with osmotic pumps into C57BL/6 mice, and pirfenidone (300 mg/kg/day) was orally administered daily for 2 wk. The lungs were removed, and single-cell suspensions were subjected to fluorescence-activated cell sorter (FACS) analysis to detect fibrocytes, which were defined as CD45 and collagen-I double-positive cells. Immunohistochemistry was performed on the lung specimens to quantify fibrocytes. Chemokines in the lung digests were measured with enzyme-linked immunosorbent assay. The effect of pirfenidone on alveolar macrophages was evaluated with bronchoalveolar lavage (BAL). In a therapeutic setting, pirfenidone administration was initiated 10 days after bleomycin treatment. For chemotaxis assay, lung fibrocytes were isolated with immunomagnetic selection (CD45-positive mesenchymal cells) after culture and allowed to migrate toward chemokines in the presence or absence of pirfenidone. Moreover, the effect of pirfenidone on the expression of chemokine receptors on fibrocytes was evaluated.
Results Pirfenidone significantly ameliorated bleomycin-induced pulmonary fibrosis as assessed with quantitative histology and collagen measurement. Fibrocyte pool size in bleomycin-treated mice lungs was attenuated from 26.5% to 13.7% by pirfenidone on FACS analysis. This outcome was also observed in a therapeutic setting. Immunohistochemistry revealed that fibrocytes were significantly decreased by pirfenidone administration compared with those in bleomycin-treated mice (P = 0.0097). Increased chemokine (CC motif) ligand-2 (CCL2) and CCL12 production in bleomycin-treated mouse lungs was significantly attenuated by pirfenidone (P = 0.0003 and P < 0.0001, respectively). Pirfenidone also attenuated macrophage counts stimulated by bleomycin in BAL fluid. Fibrocyte migration toward CCL2 and chemokine (CC motif) receptor-2 expression on fibrocytes was significantly inhibited by pirfenidone in vitro.
Conclusions Pirfenidone attenuated the fibrocyte pool size in bleomycin-treated mouse lungs via attenuation of CCL2 and CCL12 production in vivo, and fibrocyte migration was inhibited by pirfenidone in vitro. Fibrocyte inhibition is considered a mechanism of anti-fibrotic action of pirfenidone.
Introduction
Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterised by progressive scarring of the lung parenchyma.[1] Although the pathologic processes that cause disease progression are not fully understood, IPF is characterised by a microscopic pattern of usual interstitial pneumonia, which includes excessive collagen deposition, honeycombing, and the presence of fibroblastic foci.[2] Fibroblastic foci are areas of myofibroblast proliferation thought to be the main site of abnormal extracellular matrix (ECM) deposition. ECM-producing lung fibroblasts are the key source of this deposition; however, these cells are heterogeneous in a number of phenotypic features. Recent investigations have provided support for the hypothesis that they arise from several sources, including (i) resident pulmonary fibroblasts, (ii) bone marrow-derived circulating fibrocytes that infiltrate the lungs, and (iii) alveolar epithelial cells through a process called epithelial-mesenchymal transition (EMT).[3]
Among these possible sources of lung fibroblasts, bone marrow-derived circulating fibrocytes are mesenchymal progenitor cells that express markers compatible with leukocytes, hematopoietic progenitor cells, and fibroblasts.[4] Fibrocytes also express a number of other cell markers, including chemokine receptors and adhesion molecules.[5] They are chemotactically recruited to sites of tissue injury[6] and contribute to the propagation of the fibrotic response.[7] Recent reports have revealed that the number of circulating fibrocytes is significantly elevated in patients with IPF, and higher numbers are correlated with early mortality.[8] Taken together, this evidence suggests that controlling and managing fibrocytes could be a novel therapeutic approach for IPF.
Although no well-accepted medical therapy for patients with IPF has yet been established,[1] vigorous efforts to develop effective agents are being made. Among the therapeutic drugs available, pirfenidone, which has anti-fibrotic properties and was approved for the treatment of IPF in Japan in 2008, reportedly limits the decline in pulmonary function, especially that of vital capacity, that accompanies IPF.[9,10] Pirfenidone inhibits both profibrotic and proinflammatory cytokines;[11] however, its effect on fibrocytes has not been investigated.
In this study, we hypothesised that pirfenidone elicits its pharmacological effects by inhibiting fibrocytes. To test this hypothesis, we administered pirfenidone to bleomycin (BLM)-treated mice and examined the effect of pirfenidone on fibrocytes using fluorescence-activated cell sorter (FACS) analysis. We also investigated the effect of pirfenidone on chemokine production in BLM-treated mice lungs. Moreover, we examined the effect of pirfenidone on cultured fibrocyte migration and chemokine receptor expression in vitro. This study is the first to demonstrate the effect of pirfenidone on fibrocytes, which are now considered essential to the pathogenesis of IPF. Findings from our preliminary studies were reported in abstract form at a meeting of the American Thoracic Society.[12]
Respiratory Research. 2014;15(16) © 2014 BioMed Central, Ltd.
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