title: Pirfenidone inhibits fibrocyte accumulation in the lungs in bleomycin-induced murine pulmonary fibrosis.
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.
authors: Minoru
authors: M
authors: Inomata
authors: Koichiro
authors: K
authors: Kamio
authors: Department of Pulmonary Medicine and Oncology, Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan. a-azuma@nms.ac.jp.
authors: Arata
authors: A
authors: Azuma
authors: Kuniko
authors: K
authors: Matsuda
authors: Nariaki
authors: N
authors: Kokuho
authors: Yukiko
authors: Y
authors: Miura
authors: Hiroki
authors: H
authors: Hayashi
authors: Takahito
authors: T
authors: Nei
authors: Kazue
authors: K
authors: Fujita
authors: Yoshinobu
authors: Y
authors: Saito
authors: Akihiko
authors: A
authors: Gemma
mesh_terms: D000818:Animals; D001761:Bleomycin / Q000633:toxicity*; D002478:Cells, Cultured; D005260:Female; D005347:Fibroblasts / Q000187:drug effects* / Q000473:pathology; D008168:Lung / Q000187:drug effects* / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D011658:Pulmonary Fibrosis / Q000139:chemically induced* / Q000188:drug therapy* / Q000473:pathology; D011728:Pyridones / Q000494:pharmacology / Q000627:therapeutic use*
chemical_list: D011728:Pyridones; D001761:Bleomycin; C093844:pirfenidone