Tomoyuki Kakugawa

Pirfenidone attenuates expression of HSP47 in murine bleomycin-induced pulmonary fibrosis

Heat shock protein (HSP) 47, a collagen-specific molecular chaperone, is involved in the processing and/or secretion of procollagen. The present study was undertaken to investigate whether treatment with the antifibrotic drug pirfenidone attenuates the bleomycin (BL)‐induced overexpression of HSP47 in the lungs. Male ICR mice were intravenously injected with BL or saline (SA). Pirfenidone or control drug (CD) was administered 14 days after commencement of BL or SA, and continued throughout the course of the experiment. The mice were randomly divided into three experimental groups: 1) SA‐treated with CD (SA group); 2) BL‐treated with CD (BL group); and 3) BL‐treated with pirfenidone (pirfenidone group). Lungs of the pirfenidone group showed a marked reduction of fibrotic lesions compared with the corresponding BL group. Immunohistochemical studies showed that BL treatment significantly increased the number of macrophages, myofibroblasts, HSP47‐positive type II pneumocytes and HSP47‐positive interstitial spindle-shaped cells. Treatment with pirfenidone significantly reduced the number of these cells compared with the corresponding BL group. Furthermore, treatment with pirfenidone significantly suppressed the BL‐induced increase of the positive ratio of HSP47 and α‐smooth muscle actin to interstitial spindle-shaped cells. The present study results showed that pirfenidone inhibited heat shock protein 47‐positive cells and myofibroblasts, the principal cells responsible for the accumulation and deposition of extracellular matrix seen in pulmonary fibrosis.

Clinical Differences Between Interstitial Lung Disease Associated With Clinically Amyopathic Dermatomyositis and Classic Dermatomyositis

BACKGROUND Interstitial lung disease (ILD) associated with clinically amyopathic dermatomyositis (CADM) is a potentially fatal condition in which the clinical features are not well understood. The aim of the present study was to clarify the differences in clinical characteristics and prognosis of patients with ILD associated with CADM (CADM-ILD) and classic dermatomyositis associated with ILD (DM-ILD). METHODS We retrospectively studied consecutive patients with CADM-ILD and classic DM-ILD who were hospitalized between 2001 and 2007 at Nagasaki University Hospital. The study group consisted of 11 patients with CADM-ILD and 16 patients with classic DM-ILD. We compared the clinical features and prognosis between the two forms. RESULTS The Pao(2)/Fio(2) ratio was significantly lower in patients with CADM-ILD than in patients with classic DM-ILD. The lymphocyte subsets ratio in the BAL fluid of patients with CADM-ILD was significantly higher than the corresponding ratio in patients with classic DM-ILD. ILD is classified as acute or chronic, and the acute subtype was more common in patients with CADM-ILD than in those with classic DM-ILD. The mortality rate for patients with CADM-ILD (45%) was much higher than that for patients with classic DM-ILD (6%), and all of the CADM deaths occurred in the group of patients with acute CADM-ILD. CONCLUSION Our data suggest that the higher prevalence of the acute subtype of ILD in patients with CADM results in a higher mortality rate for patients with CADM-ILD.

Pirfenidone inhibits TGF-β1-induced over-expression of collagen type I and heat shock protein 47 in A549 cells

BackgroundPirfenidone is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis (IPF). We previously showed that pirfenidone inhibits the over-expression of collagen type I and of heat shock protein (HSP) 47, a collagen-specific molecular chaperone, in human lung fibroblasts stimulated with transforming growth factor (TGF)-β1 in vitro. The increased numbers of HSP47-positive type II pneumocytes as well as fibroblasts were also diminished by pirfenidone in an animal model of pulmonary fibrosis induced by bleomycin. The present study evaluates the effects of pirfenidone on collagen type I and HSP47 expression in the human alveolar epithelial cell line, A549 cells in vitro.MethodsThe expression of collagen type I, HSP47 and E-cadherin mRNAs in A549 cells stimulated with TGF-β1 was evaluated by Northern blotting or real-time PCR. The expression of collagen type I, HSP47 and fibronectin proteins was assessed by immunocytochemical staining.ResultsTGF-β1 stimulated collagen type I and HSP47 mRNA and protein expression in A549 cells, and pirfenidone significantly inhibited this process. Pirfenidone also inhibited over-expression of the fibroblast phenotypic marker fibronectin in A549 cells induced by TGF-β1.ConclusionWe concluded that the anti-fibrotic effects of pirfenidone might be mediated not only through the direct inhibition of collagen type I expression but also through the inhibition of HSP47 expression in alveolar epithelial cells, which results in reduced collagen synthesis in lung fibrosis. Furthermore, pirfenidone might partially inhibit the epithelial-mesenchymal transition.

Direct hemoperfusion using immobilized polymyxin B in patients with rapidly progressive interstitial pneumonias: a retrospective study.

Background: Rapidly progressive interstitial pneumonia (IP), including acute exacerbation of IP, has a high mortality rate. Direct hemoperfusion with a polymyxin B-immobilized fiber column (PMX-DHP) was recently identified as an effective treatment for sepsis-associated acute respiratory distress syndrome. However, little is known about the effectiveness of PMX-DHP for rapidly progressive IP. Objectives: The present study investigates whether PMX-DHP is safe and effective against rapidly progressive IP. Methods: We retrospectively examined the effects of PMX-DHP in 33 consecutive patients with rapidly progressive IP who were resistant to steroid pulse therapy. Patients were hospitalized at Nagasaki University Hospital between 2006 and 2009. Results: Seventy-two hours after PMX-DHP, the arterial oxygen tension/inspiratory oxygen fraction ratio (median 127–153 mm Hg) had significantly improved. One week after PMX-DHP, the arterial oxygen tension/inspiratory oxygen fraction ratio (median 127–227 mm Hg), the alveolar-arterial difference of oxygen (median 371–177 mm Hg) and the number of positive criteria for systemic inflammatory response syndrome had significantly improved, despite the ineffectiveness of corticosteroid pulse therapy. The serum level of monocyte chemotactic protein 1 was significantly decreased immediately after PMX-DHP. Conclusions: PMX-DHP was safe and effective in improving oxygenation and systemic inflammatory response syndromein patients with rapidly progressive IP. The beneficialeffects of PMX-DHP may be at least partially due to the inhibition of monocyte activation.

Expression of HSP47 in Usual Interstitial Pneumonia and Nonspecific Interstitial Pneumonia

BackgroundHeat shock protein (HSP) 47, a collagen-specific molecular chaperone, is involved in the processing and/or secretion of procollagens, and its expression is increased in various fibrotic diseases. The aim of this study was to determine whether quantitative immunohistochemical evaluation of the expression levels of HSP47, type I procollagen and α-smooth muscle actin (SMA) allows the differentiation of idiopathic usual interstitial pneumonia (UIP) from UIP associated with collagen vascular disease (CVD) and idiopathic nonspecific interstitial pneumonia (NSIP).MethodsWe reviewed surgical lung biopsy specimens of 19 patients with idiopathic UIP, 7 with CVD-associated UIP and 16 with idiopathic NSIP and assigned a score for the expression of HSP47, type I procollagen and α-SMA in type II pneumocytes and/or lung fibroblasts (score 0 = no; 1 = weak; 2 = moderate; 3 = strong staining).ResultsThe expression level of HSP47 in type II pneumocytes of idiopathic UIP was significantly higher than in CVD-associated UIP and idiopathic NSIP. The expression of HSP47 in fibroblasts was significantly higher in idiopathic UIP and idiopathic NSIP than in CVD-associated UIP. The expression of type I procollagen in type II pneumocytes was significantly higher in idiopathic UIP than in idiopathic NSIP. The expression of type I procollagen in fibroblasts was not different in the three groups, while the expression of α-SMA in fibroblasts was significantly higher in idiopathic UIP than in idiopathic NSIP.ConclusionOur results suggest the existence of different fibrotic pathways among these groups involved in the expression of HSP47 and type I procollagen.

Elevated levels of interferon γ‐inducible protein‐10 and epithelial neutrophil‐activating peptide‐78 in patients with pulmonary sarcoidosis

Objective and background:  Interferon γ‐inducible protein (IP)‐10 and epithelial neutrophil‐activating peptide (ENA)‐78 belong to the CXC chemokine family and are important factors in inflammatory lung diseases. In sarcoidosis, the potential role of IP‐10 to regulate the migration and activation of T‐cells towards sites of sarcoid activity has been suggested.

S100A9 in BALF is a candidate biomarker of idiopathic pulmonary fibrosis

BACKGROUND Idiopathic pulmonary fibrosis (IPF) is a specific form of chronic, progressive fibrosing interstitial pneumonia of unknown cause, and the prognosis remains poor. On the other hand, other fibrotic interstitial pneumonias such as idiopathic nonspecific interstitial pneumonia (I-NSIP) and collagen vascular disease-associated interstitial pneumonia (CVD-IP) resemble IPF, but they respond to therapy and the prognosis is better. We searched for biomarkers to distinguish IPF from other fibrotic interstitial pneumonias and investigated whether S100A9 could be useful for discriminating types of fibrotic interstitial pneumonia based on our preliminary proteomic findings. METHODS We measured S100A9 levels in serum and bronchoalveolar lavage fluid (BALF) from 28 patients with IPF, 15 with I-NSIP, 20 with cryptogenic organizing pneumonia (COP), 35 with CVD-IP and 23 healthy individuals (controls) using enzyme-linked immunosorbent assays. S100A9 in the lung was also immunohistochemically localized. RESULTS S100A9 levels in BALF, but not in serum, were significantly elevated in patients with IPF compared with I-NSIP, COP, CVD-IP and healthy individuals. S100A9 immunoreactivity was localized mainly in macrophages and neutrophils in lung specimens from patients with IPF. The results of receiver operating characteristic (ROC) curve analysis showed that BALF S100A9 levels had sufficient specificity and sensitivity to distinguish IPF from I-NSIP and CVD-IP. CONCLUSION S100A9 in BALF might serve as a candidate biomarker to discriminate between IPF and other fibrotic interstitial pneumonias.

Serum interferon-α is a useful biomarker in patients with anti-melanoma differentiation-associated gene 5 (MDA5) antibody-positive dermatomyositis

Abstract Objective. We have tried to clarify the clinical importance of the measurement of serum type-I interferon (IFN) in patients with anti-melanoma differentiation-associated gene 5 Ab (MDA5 Ab)-positive dermatomyositis (DM). Methods. We studied 30 patients with DM: 10 were anti-MDA5 Ab-positive and 20 were anti-MDA5 Ab-negative. At each patients initial visit, serum IFN-α, IFN-β, interleukin 18 (IL-18), ferritin, and the titer of anti-MDA5 Ab were measured using enzyme-linked immunosorbent assays (ELISAs). The associations between the IFNs and with the other variables were examined. Results. Rapidly progressive interstitial lung disease (RPILD) was confirmed in 10 patients, most of whom were complicated in the anti-MDA5 Ab-positive DM patients. The presence of clinically amyopathic dermatomyositis (CADM) as well as the serum concentrations of IFN-α and ferritin was significantly higher in the anti-MDA5 Ab-positive DM patients. Serum concentration of IL-18 did not differ between anti-MDA5 Ab-positive and anti-MDA5 Ab-negative groups; however, a positive correlation was found between IFN-α and IL-18 in the anti-MDA5 Ab-positive DM patients (r = 0.8139, p = 0.0146). Conclusion. Serum IFN-α can be used as a useful biomarker in patients with anti-MDA5 Ab-positive DM, which may reflect the presence of RPILD.

Differential effects of human neutrophil peptide-1 on growth factor and interleukin-8 production by human lung fibroblasts and epithelial cells

ABSTRACT α-Defensins, antimicrobial peptides produced mainly by neutrophils, have been reported to be associated with a wide variety of lung diseases, including idiopathic pulmonary fibrosis (IPF), cystic fibrosis (CF), and diffuse panbronchiolitis (DPB). In each disease, α-defensins are located in different areas, such as around the alveolar septa in IPF and around the airways in CF and DPB, suggesting that α-defensins play different roles. Meanwhile, growth factors are known to contribute to IPF, CF, and DPB. α-Defensins are known to induce interleukin (IL)-8 in airway epithelial cells, but the effects of α-defensins on the release of growth factors from various components in the lung have not been sufficiently investigated. In the present study, the in vitro effects of human neutrophil peptide (HNP)-1 (a subtype of α-defensin) on the expressions of IL-8 and growth factors in lung fibroblasts, bronchial epithelial cells, and alveolar epithelial cells were examined. HNP-1 mainly enhanced the expression of IL-8 in epithelial cells, whereas it enhanced transforming growth factor-β and vascular endothelial growth factor expressions in lung fibroblasts. These results suggest that α-defensins play different roles in the pathogenesis of IPF, CF, and DPB according to the location in the lung where the α-defensins are mainly produced.

Effects of Doxycycline on Production of Growth Factors and Matrix Metalloproteinases in Pulmonary Fibrosis

Background: Idiopathic pulmonary fibrosis (IPF) is characterized by progressive fibrosis and a poor prognosis. Alveolar epithelial cells (AECs) are considered to play important roles by releasing growth factors and matrix metalloproteinases (MMPs) and by being involved in epithelial mesenchymal transition in IPF. Doxycycline hydrochloride (DOXY), an inhibitor of MMPs, attenuates pulmonary fibrosis in models and in patients with IPF; however, the mechanism of this action remains obscure. Objectives: The present study investigated the effect of DOXY on growth factors and MMP production in AECs. Methods: Bleomycin (BL)-induced murine pulmonary fibrosis was treated with DOXY and examined by pathological and immunohistochemical staining. The human alveolar epithelial cell line A549 was stimulated with transforming growth factor (TGF)-β1 and incubated with DOXY, and then the expression of growth factors, MMPs, and collagen type I was evaluated at the mRNA and protein levels. We also evaluated the effects of DOXY on the TGF-β1-induced Smad signaling pathway. Results: DOXY reduced fibrosis scores and the production of collagen type I, connective tissue growth factor (CTGF), and TGF-β1 in BL models. DOXY inhibited the mRNA expression of MMP-2, MPP-9, CTGF, and collagen type I as well as the production of MMP-2 and platelet-derived growth factor-AA protein induced in A549 cells by TGF-β1 but not by Smad2 and Smad3 phosphorylation. We did not find a similar effect of DOXY in normal lung fibroblasts. Conclusions: Our results suggest that DOXY could be useful for attenuating pulmonary fibrosis through the inhibition of growth factors and MMP production in AECs.