Time course of bleomycin‐induced lung fibrosis

Intratracheal instillation (IT) of bleomycin is a widely used experimental model for lung fibrosis. In this study we describe the time‐course of bleomycin‐induced lung fibrosis in mice using computer‐assisted morphometry. C57Bl/6J mice were treated with a single IT dose of bleomycin or control saline. Animals were killed 3, 6, 14 and 21 days post‐IT. Lung injury was evaluated by analysis of bronchoalveolar lavage (BAL) fluid, hydroxyproline concentration in the lung, routine light microscopic examination resulting in a semiquantitative morphological index (SMI) of lung injury, and quantitative morphological measurements (fibrosis fraction and alveolar wall area fraction) aided by optimas image analysis software. Changes in BAL fluid attributed to bleomycin treatment include increased total cell count (days 14 and 21), and increased percentage of neutrophils (days 3 and 6) followed by a sustained increase in lymphocytes (days 6, 14 and 21). Hydroxyproline levels increased in bleomycin‐treated mice on days 14 and 21. Median SMI grades were significantly elevated on days 3, 14 and 21. Computer‐assisted morphometry demonstrated a 3‐fold increase in fibrosis fraction and a 1.3‐fold increase in wall area fraction in bleomycin‐treated mice on day 14, with no further increase on day 21. These data also demonstrate that the most suitable time point for assessing lung fibrosis in this model is 14 days after IT instillation of bleomycin, based on the observation that at 14 days the animals developed extensive fibrosis, but had less variability in the fibrotic response and lower mortality than later at 21 days. Computer‐assisted morphometry provides objective and quantitative measurements that are a useful tool for the evaluation of bleomycin‐induced lung injury.

[1]  G. W. Snedecor.  Statistical Methods , 1964 .

[2]  J. R. Baker,et al.  Bleomycin-induced interstitial pneumonia in dogs , 1971, Thorax.

[3]  J. Brentjens,et al.  EXPERIMENTAL IMMUNE COMPLEX DISEASE OF THE LUNG THE PATHOGENESIS OF A LABORATORY MODEL RESEMBLING CERTAIN HUMAN INTERSTITIAL LUNG DISEASES , 1974 .

[4]  R. Johnson,et al.  Lymphokine stimulation of collagen accumulation. , 1976, The Journal of clinical investigation.

[5]  W. Roberts,et al.  Analysis of cellular and protein content of broncho-alveolar lavage fluid from patients with idiopathic pulmonary fibrosis and chronic hypersensitivity pneumonitis. , 1977, The Journal of clinical investigation.

[6]  B. Celli,et al.  Chronic interstitial pulmonary fibrosis produced in hamsters by endotracheal bleomycin. Lung volumes, volume-pressure relations, carbon monoxide uptake, and arterial blood gas studied. , 1978, The American review of respiratory disease.

[7]  M. Schwarz,et al.  Immunofluorescent patterns in the idiopathic interstitial pneumonias. , 1978, The Journal of laboratory and clinical medicine.

[8]  R. Crystal,et al.  Bleomycin-induced interstitial pulmonary disease in the nude, athymic mouse. , 2015, The American review of respiratory disease.

[9]  R. Thrall,et al.  The effect of antilymphocyte globulin on the development of bleomycin-induced pulmonary fibrosis in the rat , 1979 .

[10]  D. Simmons,et al.  Diffuse pulmonary interstitial disease; an immunohistologic study. , 1979, Chest.

[11]  E. Holborow,et al.  Circulating immune complexes in patients with cryptogenic fibrosing alveolitis. , 1979, Clinical and experimental immunology.

[12]  R. Thrall,et al.  Bleomycin-induced pulmonary fibrosis. Effects of steroid on lung collagen metabolism. , 1981, The American review of respiratory disease.

[13]  J. Seyer,et al.  Alterations in collagen production in mixed mononuclear leukocyte- fibroblast cultures , 1983, The Journal of experimental medicine.

[14]  S. Phan,et al.  The effects of the nude (nu/nu) mutation on bleomycin-induced pulmonary fibrosis. A biochemical evaluation. , 1983, The American review of respiratory disease.

[15]  S. Wahl,et al.  Modulation of fibroblast growth by a lymphokine of human T cell continuous T cell line origin. , 1983, Journal of immunology.

[16]  D. Hyde,et al.  Morphometric estimates of infiltrative cellular changes during the development of bleomycin-induced pulmonary fibrosis in hamsters. , 1983, The American journal of pathology.

[17]  Clive R. Taylor,et al.  Immune complexes, gallium lung scans, and bronchoalveolar lavage in idiopathic interstitial pneumonitis-fibrosis. , 1983 .

[18]  W. Border,et al.  Immune complexes, gallium lung scans, and bronchoalveolar lavage in idiopathic interstitial pneumonitis-fibrosis. , 1983, Chest.

[19]  R. Thrall,et al.  A comparison of lymphocyte populations in lung tissue and in bronchoalveolar lavage fluid of rats at various times during the development of bleomycin-induced pulmonary fibrosis. , 1984, The American review of respiratory disease.

[20]  R. K. Kumar,et al.  Separation and characterization of lymphocytes from rat lung parenchyma. , 1984, Experimental lung research.

[21]  J. Seyer,et al.  Lymphocyte modulation of fibroblast function in vitro: stimulation and inhibition of collagen production by different effector molecules. , 1984, Journal of immunology.

[22]  T. Takita,et al.  Immunological involvement in pulmonary fibrosis induced by peplomycin. , 1985, The Journal of antibiotics.

[23]  E. Frome,et al.  Intratracheal versus intravenous administration of bleomycin in mice: acute effects. , 1986, Toxicology and applied pharmacology.

[24]  J. Lazo,et al.  High dose continuous infusion of bleomycin in mice: a new model for drug-induced pulmonary fibrosis. , 1987, The Journal of pharmacology and experimental therapeutics.

[25]  E. Brianti,et al.  Study of immune complexes in bronchoalveolar lavage fluids. , 1988, Respiration; international review of thoracic diseases.

[26]  M. Hacker,et al.  Effect of cytotoxic monoclonal antibody depletion of T-lymphocyte subpopulations on bleomycin-induced lung damage in C57BL/6J mice. , 1989, Toxicology and applied pharmacology.

[27]  R. Thrall,et al.  Superoxide production by rat neutrophils in the oleic acid model of lung injury. , 1990, Free radical biology & medicine.

[28]  Y. Endo,et al.  A Study of Human Interstitial Lung Diseases with Special Reference to Immune Complexes and Hyaline Membrane , 1990, Acta pathologica japonica.

[29]  R. Harbeck,et al.  Class II antigens of the major histocompatibility complex are increased in lungs of bleomycin-treated rats. , 1990, Immunology letters.

[30]  S. Sharma,et al.  The effect of an anti-CD3 monoclonal antibody on bleomycin-induced lymphokine production and lung injury. , 1996, American journal of respiratory and critical care medicine.

[31]  D. Lamb,et al.  THE IMMUNOLOGICAL ARCHITECTURE OF B‐LYMPHOCYTE AGGREGATES IN CRYPTOGENIC FIBROSING ALVEOLITIS , 1996, The Journal of pathology.

[32]  J. Myers,et al.  Idiopathic pulmonary fibrosis: clinical relevance of pathologic classification. , 1998, American journal of respiratory and critical care medicine.

[33]  R. Swiderski,et al.  Differential expression of extracellular matrix remodeling genes in a murine model of bleomycin-induced pulmonary fibrosis. , 1998, The American journal of pathology.

[34]  I. Lossos,et al.  Effect of Immunomodulators on Bleomycin-Induced Lung Injury , 1999, Respiration.

[35]  I. Lossos,et al.  The effect of enoxaparin on bleomycin-induced lung injury in mice. , 1999, Experimental lung research.

[36]  R. Ueda,et al.  Elevation of Anti-Cytokeratin 18 Antibody and Circulating Cytokeratin 18: Anti-Cytokeratin 18 Antibody Immune Complexes in Sera of Patients with Idiopathic Pulmonary Fibrosis , 2000, Lung.

[37]  I. Lossos,et al.  The effect of suramin on bleomycin-induced lung injury. , 2000, Life sciences.

[38]  J. Fujita,et al.  Circulating Cytokeratin 8:Anti-Cytokeratin 8 Antibody Immune Complexes in Sera of Patients with Pulmonary Fibrosis , 2000, Respiration.

[39]  G. Hunninghake,et al.  Idiopathic pulmonary fibrosis. , 2001, The New England journal of medicine.

[40]  I. Lossos,et al.  Human Recombinant Interferon-α2a and Interferon-αA/D Have Different Effects on Bleomycin-Induced Lung Injury , 2001, Respiration.

[41]  J. Coalson.  The ultrastructure of human fibrosing alveolitis , 2004, Virchows Archiv A.

[42]  R. Thrall,et al.  Superoxide anion production by rat neutrophils at various stages of bleomycin-induced lung injury , 2004, Lung.