Rationale: Fibroblasts are believed to be the major cells responsible for the production and maintenance of extracellular matrix. Alterations in fibroblast functional capacity, therefore, could play a role in the pathogenesis of pulmonary emphysema, which is characterized by inadequate maintenance of tissue structure. Objectives: To evaluate the hypothesis that deficient fibroblast repair characterizes cells obtained from individuals with chronic obstructive pulmonary disease (COPD) compared with control subjects. Methods: Fibroblasts were cultured from lung tissue obtained from individuals undergoing thoracotomy and were characterized in vitro. Measurements and Main Results: Fibroblasts from individuals with COPD, defined by reduced FEV 1 , manifested reduced chemotaxis toward fibronectin and reduced contraction of three-dimensional collagen gels, two bioassays associated with fibroblast repair function. At least two mechanisms appear to account for these differences. Prostaglandin E (PGE), a known inhibitor of fibroblast repair functions, was produced in increased amount by fibroblasts from subjects with COPD, which also expressed increased amounts of the receptors EP2 and EP4, both of which signal through cyclic AMP. Incubation of fibroblasts with indomethacin or with the PKA inhibitor KT-5720 partially restored COPD subject fibroblast function. In addition, fibroblasts from subjects with COPD produced more transforming growth factor (TGF)-b1, but manifested reduced response to TGF-b1. The functional alterations in fibroblasts correlated with both lung function assessed by FEV 1 and, for the data available, with severity of emphysema assessed by DL CO . Conclusions: Fibroblasts from individuals with COPD have reduced capability to sustain tissue repair, which suggests that this may be one mechanism that contributes to the development of emphysema.

BackgroundBone 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.MethodsBleomycin (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.ResultsPirfenidone 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.ConclusionsPirfenidone 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.

Diffuse panbronchiolitis (DPB) is a chronic inflammatory airway disease predominantly affecting Asian populations. DPB is considered to be a complex genetic disease. Considering the mucous hypersecretion of the disease, we hypothesized that the transcriptional activity of mucin genes may be altered in DPB. We analyzed nucleotide sequences of regulatory region of six mucin genes--MUC1, MUC2, MUC4, MUC5AC, MUC5B, and MUC7--and detected their promoter polymorphisms. Among them, the insertion/deletion polymorphism identified in the MUC5B gene was significantly associated with the disease (p = 0.0001). Transcriptional activity observed in the three major promoter haplotypes corresponded to the strength of the disease association in which these haplotypes are involved. Immunohistochemistry of the lung tissues of DPB revealed that MUC5B was abundantly expressed not only in bronchial glands but also in increased numbers of goblet cells on the bronchial surface, where MUC5AC is predominant and MUC5B expression is generally scarce in the normal lung. Marked mucous hypersecretion observed in DPB may be partly explained by increased and aberrant expression of MUC5B. The possible involvement of MUC5B gene in DPB was demonstrated. A further role of the MUC5B polymorphism in its pathogenesis should be studied in the future.

Fibrotic diseases are characterized by the accumulation of extracellular matrix together with distortion and disruption of tissue architecture. Phosphodiesterase (PDE)4 inhibitors, by preventing the breakdown of cAMP, can inhibit fibroblast functions and may be able to mitigate tissue remodeling. Transforming growth factor (TGF)-beta1, a mediator of fibrosis, can potentially modulate cAMP by altering PGE(2) metabolism. The present study assessed whether PDE4 inhibitors functionally antagonize the profibrotic activity of fibroblasts stimulated by TGF-beta1. The PDE4 inhibitors roflumilast and rolipram both inhibited fibroblast-mediated contraction of three-dimensional collagen gels and fibroblast chemotaxis toward fibronectin in the widely studied human fetal lung fibroblast strain HFL-1 and several strains of fibroblasts from adult human lung. Roflumilast was approximately 10-fold more potent than rolipram. There was a trend for PDE4 inhibitors to inhibit more in the presence of TGF-beta1 (0.05 < P < 0.08). The effect of the PDE4 inhibitors was mediated through cAMP-stimulated protein kinase A (PKA), although a PKA-independent effect on gel contraction was also observed. The effect of PDE4 inhibitors depended on fibroblast production of PGE(2) and TGF-beta1-induced PGE(2) production. PDE4 inhibitors together with TGF-beta1 resulted in augmented PGE(2) production together with increased expression of COX mRNA and protein. The present study supports the concept that PDE4 inhibitors may attenuate fibroblast activities that can lead to fibrosis and that PDE4 inhibitors may be particularly effective in the presence of TGF-beta1-induced fibroblast stimulation.

Background/Aim: Recent reports suggest that polymyxin B (PMX)-immobilized fiber may have beneficial effects in idiopathic pulmonary fibrosis (IPF) with acute exacerbation (AE). High mobility group box-1 (HMGB-1) is an important pro-inflammatory mediator that contributes to acute lung inflammation. This study was aimed to investigate whether PMX treatment affects serum HMGB-1 levels and oxygenation in IPF patients with AE. Materials and Methods: Twenty IPF patients with AE were treated by PMX. PMX treatment was carried out once daily for 2 successive days. Serum HMGB-1 levels were measured before and after PMX treatment. We also monitored arterial oxygen tension (PaO₂)/inspiratory oxygen fraction (FiO₂) (P/F) ratio. PMX fiber columns were analyzed to examine whether HMGB-1 was absorbed by PMX. Results: PMX treatment significantly improved both the serum HMGB-1 level and P/F ratio. HMGB-1 was detected in washing medium from the PMX column. Conclusion: PMX treatment may reduce serum HMGB-1 and improve oxygenation in patients with IPF with AE.

Reactive nitrogen species (RNS) such as peroxynitrite cause cellular injury and tissue inflammation. Excessive production of nitrotyrosine, which is a footprint of RNS, has been observed in the airways of patients with asthma and chronic obstructive pulmonary disease, disorders characterized by tissue remodeling. The aim of this study was to evaluate whether RNS can affect tissue remodeling through direct effects on fibroblasts, and to determine if these effects depend on production of transforming growth factor-beta (TGF-beta). To accomplish this, human fetal lung fibroblasts (HFL-1) were used to assess fibroblast-mediated contraction of floating gels and chemotaxis toward fibronectin. In addition, the ability of fibroblasts to release TGF-beta1, fibronectin, and vascular endothelial growth factor (VEGF) was assessed by enzyme-linked immunosorbent assay. Authentic peroxynitrite significantly augmented gel contraction (P < 0.01) and chemotaxis (P < 0.01) compared with control in a concentration-dependent manner. Similarly, the peroxynitrite donor 3-morpholynosidenonimine hydrochloride (SIN-1) also augmented gel contraction (P < 0.01). RNS also significantly increased TGF-beta1 (P < 0.01), fibronectin (P < 0.01), and VEGF (P < 0.01) release into the media in both 3D gel and monolayer culture. Anti-TGF-beta antibody reversed RNS-augmented gel contraction (P < 0.01) and mediator production (P < 0.01). Anti-TGF-beta antibody also partially, but significantly, reversed RNS-augmented chemotaxis toward fibronectin (P < 0.01). Finally, peroxynitrite enhanced expression of alpha5beta1 integrin, which is a receptor for fibronectin (P < 0.01), and neutralizing anti-TGF-beta antibody suppressed peroxynitrite-augmented alpha5beta1 expression (P < 0.01). These results suggest that RNS can affect the tissue repair process by modulating TGF-beta1.

Asthmatic airway remodeling is characterized by goblet cell hyperplasia, angiogenesis, smooth muscle hypertrophy, and subepithelial fibrosis. This study evaluated whether acquired changes in fibroblast phenotype could contribute to this remodeling. Airway and parenchymal fibroblasts from control or chronically ovalbumin (OVA)-sensitized and challenged ''asthmatic'' mice were assessed for several functions related to repair and remodeling 6 exogenous transforming growth factor (TGF)-b. All OVA-challenged mouse fibroblasts demonstrated augmented gel contraction (P , 0.05) and chemotaxis (P , 0.05); increased TGF-b 1 (P , 0.05), fibronectin (P , 0.05), and vascular endothelial growth factor (P , 0.05) release; and expressed more a-smooth muscle actin (P , 0.05). TGF-b 1 stimulated both control and asthmatic fibroblasts, which retained all differences from control fibroblasts for all features(P , 0.05, all comparisons). Parenchymal fibroblasts proliferated more rapidly (P , 0.05), while airway fibroblasts proliferated similarly compared with control fibroblasts (P 5 0.25). Thus, in this animal model, OVAchallenged mouse fibroblasts acquire a distinct phenotype that differs from control fibroblasts. The augmented profibrotic activity and mediator release of asthmatic fibroblasts could contribute to airway remodeling in asthma.

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