, Volume 24, Issue 5, pp 477–491 | Cite as

Anti-Inflammatory Effect of Pirfenidone in the Bleomycin-Hamster Model of Lung Inflammation

  • Swarnalatha N. Iyer
  • Dallas M. Hyde
  • Shri N. Giri


We have previously reported the antifibrotic effects of pirfenidone (PD) in the bleomycin (BL)-hamster model of lung fibrosis. Since the development of fibrosis is generally preceded by acute lung inflammation, the present study was conducted to find out if dietary intake of PD (0.5%) has any effects on BL-induced lung inflammation. In this regard, we evaluated the effects of PD on BL-induced increased pulmonary vascular permeability, increased influx of inflammatory cells and increased levels of TGF-β in the bronchoalveolar lavage fluid (BALF). Hamsters were intratracheally (IT) instilled with saline (SA) or BL (5.5 units/kg/5 ml). The animals were fed the control diet (CD) or the same diet containing 0.5% PD 2 days prior to IT instillation and throughout the study. The bronchoalveolar lavage was carried out at different times after IT instillation. Lavage fluid was used for total and differential cell counts and BALF-supernatant for measurement of total protein and TGF-β. IT instillation of BL caused significant increases in total cells, neutrophils, macrophages and lymphocytes and in the levels of total protein and TGF-β in BALF from hamsters in the BL + CD groups as compared to the corresponding SA + CD control groups. In contrast, treatment with pirfenidone in general, suppressed the BL-induced increases in the levels of proteins and TGF-β and in the influx of neutrophils, macrophages and lymphocytes in BALF at the early time points in BL + PD groups. Based on the data reported in this study, we conclude that the anti-inflammatory effects of pirfenidone as evident by suppressions of BL-induced increased pulmonary vascular permeability and increased influx of inflammatory cells in the lung contribute additionally to its inherent anti-fibrotic effect.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    RENNARD, S. I., P. B. BITTERMAN, AND R. G. CRYSTAL. 1984. Current concepts of the pathogenesis of Fibrosis: Lessons from Pulmonary fibrosis. Prog. Clin. Biol. Res. 154:359-377.Google Scholar
  2. 2.
    KUMAR, R. K. and T. W. J. LYKE. 1995. Messages and Handshakes: Cellular interaction in pulmonary fibrosis. Pathol. 27:18-26.Google Scholar
  3. 3.
    RYAN, G. B. and G. MAJNO. 1977. Chronic Inflammation, Inflammation, The Upjohn Co, 58-59.Google Scholar
  4. 4.
    THRALL, R. S. and P. J. SCALISE. 1995. Bleomycin. In: Pulmonary fibrosis. S. M. Phan, R. S. Thrall, editors. Marcel Dekker, Inc., New York. 80:241-242.Google Scholar
  5. 5.
    LAZO, J. S., D. G. HYOTT, S. M. SEBTI, and B. R. PITT. (1990). Bleomycin: A pharmacologic tool in the study of the pathogenesis of interstitial pulmonary fibrosis. Pharmacol. Ther. 47:347-358.Google Scholar
  6. 6.
    WEINBERGER, S. E., J. A. KELMAN, N. A. ELSON, R. C. J. YOUNG, H. Y. REYNOLDS, J. D. FULLMER, and R. G. CRYSTAL. (1978). Bronchoalveolar lavage in interstitial lung disease. Ann. Int. Med. 89:459-466.Google Scholar
  7. 7.
    MARTIN, W. J., J. E. GADEK, G. W. HUNNINGHAKE, et al. 1981. Oxidant injury of lung parenchymal cells. J. Clin. Invest. 68:1277-1288.Google Scholar
  8. 8.
    HUNNINGHAKE, G. W., J. E. GADEK, O. KAWANAMI, V. S. FERRANS, R. J. CRYSTAL. 1979. Inflammatory and immune processes in the human lung in health and disease: Evaluation by bronchoalveolar lavage. Am. J. Pathol. 97:149-206.Google Scholar
  9. 9.
    TURNER-WARWICK, M. and P. L. HASLAM. 1987. The value of serial bronchoalveolar lavage in assessing the clinical progress of patients with cryptogenic fibrosing alveolitis. Am. Rev. Respir. Dis. 135:26-34.Google Scholar
  10. 10.
    SNIDER, G., B. R. CELLI, R. H. GOLDSTEIN, J. J. O'BRIEN, and E. C. LUCEY. 1978. Chronic interstitial pulmonary fibrosis produced in hamsters by endotracheal bleomycin. Am. Rev. Respir. Dis. 117:289-297.Google Scholar
  11. 11.
    WHITE, D. A., M. G. KRIS, and D. E. STOVER. 1987. Bronchoalveolar lavage cell populations in bleomycin lung toxicity. Thorax 42:551-552.Google Scholar
  12. 12.
    THRALL, R. S., R. W. BARTON, D'AMATO, and S. B. SULAVIK. 1982. Differential cellular analysis of bronchoalveolar lavage fluid obtained at various stages during the development of bleomycin-induced pulmonary fibrosis in the rat. Am. Rev. Respir. Dis. 126:488-492.Google Scholar
  13. 13.
    HUNNINGHAKE, G. W., J. E. GADEK, H. M. FALES, and R. G. CRYSTAL. 1980. Human alveolar macrophage-derived chemotactic factor for neutrophils. Stimuli and partial characterization. J. Clin. Invest. 66:473-483.Google Scholar
  14. 14.
    ZHANG, Y., T. C. LEE, and B. GUILLEMIN. 1993. Enhanced IL-1β and TNF-α release and messenger RNA expression in macrophages from idiopathic pulmonary fibrosis or after asbestos exposure. J. Immunol. 150:4188-4196.Google Scholar
  15. 15.
    VANHEE, D., P. GOSSET, and B. WALLERT. 1994. Mechanisms of fibrosis in coal workers pneumoconiosis: increased production of platelet-derived growth factor, insulin-like growth factor type I and transforming growth factor-α and relationship to disease severity. Am. J. Respir. Crit. Care Med. 150:1049-1055.Google Scholar
  16. 16.
    SHAW, R. J., S. H. BENEDICT, and R. A. F. CLARK. 1991. Pathogenesis of pulmonary fibrosis in interstitial lung disease: alveolar macrophage PDGF (β) gene activation and up-regulation by interferon gamma. Am. Rev. Respir. Dis. 143:163-173.Google Scholar
  17. 17.
    KHALIL, N. and R. O'CONNOR. 1995. Cytokine regulation of pulmonary fibrosis: Transforming growth factor-α. In: Pulmonary fibrosis. S. M. Phan, R. S. Thrall, editors. Marcel Dekker, Inc., New York. 80:627-641.Google Scholar
  18. 18.
    KHALIL, N., O. BERENZAY, M. B. SPORN, and A. H. GREENBERG. 1989. Macrophage production of transforming growth factor-α and fibroblast collagen synthesis in chronic pulmonary inflammation. J. Exp. Med. 170:727-737.Google Scholar
  19. 19.
    KHALIL, N., S. CORNE, C. WHITMAN, and H. YACYSHYN. 1996. Plasmin regulates the activation of cell-associated TGF-β secreted by rat alveolar macrophages after in vivo bleomycin injury. Am. J. Respir. Cell Mol. Biol. 15:252-259.Google Scholar
  20. 20.
    IGNOTZ, R. A. and J. MASSAGUE. 1986. Transforming growth factor-b stimulates the expression of fibronectin and collagen and their incorporation into extracellular matrix. J. Biol. Chem. 261:4335-4345.Google Scholar
  21. 21.
    IYER, S. N., J. S. WILD, M. J. SCHIEDT, D. M. HYDE, S. B. MARGOLIN, and S. N. GIRI. 1995. Dietary intake of pirfenidone ameliorates bleomycin induced lung fibrosis in hamsters. J. Lab. Clin. Med. 125:779-785.Google Scholar
  22. 22.
    IYER, S. N., S. B. MARGOLIN, D. M. HYDE, and S. N. GIRI. 1998. Lung Fibrosis is ameliorated by pirfenidone fed in diet after the second dose in a three dose bleomycin hamster model. Exp. Lung. Res. 24:119-132.Google Scholar
  23. 23.
    IYER, S. N., G. GURUJEYALAKSHMI, and S. N. GIRI. 1999. Downregulation of procollagen genes at the transcriptional level in the bleomycin-induced model of lung fibrosis. J. Pharm. Exp. Ther. 289:211-218.Google Scholar
  24. 24.
    WILCOX, M. L., A. KERVITSKY, L. C. WATTERS, and T. E. KING. 1988. Quantification of cells recovered from bronchoalveolar lavage: comparison of cytocentrifuge preparations with the filter method. Am. Rev. Respir. Dis. 158:74-80.Google Scholar
  25. 25.
    LOWRY, O. H., N. J. ROSEBROUGH, A. L. FARR, and R. J. RANDALL. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275.Google Scholar
  26. 26.
    CRYSTAL, R. G., P. B. BITTERMAN, S. I. RENNARD, A. J. HANCE, and B. A. KEOGH. 1984. Interstitial lung diseases of unknown cause. Disorders characterized by chronic inflammation of the lower respiratory tract (first of two parts). New Engl. J. Med. 310:154-166.Google Scholar
  27. 27.
    GIRI, S. N. and Q. WANG. 1995. Miscellaneous mediators systems. In: Pulmonary fibrosis. S. M. Phan, R. S. Thrall, editors. Marcel Dekker, Inc., New York. 80:777-836.Google Scholar
  28. 28.
    SMITH, R. E., R. M. STRIETER, S. M. PHAN, et al. 1996. C-C Chemokines: Novel mediators of the profibrotic inflammatory response to bleomycin challenge. Am. J. Respir. Cell Mol. Biol. 15:693-702.Google Scholar
  29. 29.
    GIRI, S. N., M. A. HOLLINGER, and M. J. SCHIEDT. 1981. The effects of paraquat and superoxide dismutase on pulmonary vascular permeability and edema in mice. Arch. Environ. Health 36:149-154.Google Scholar
  30. 30.
    ASO, Y., K. YONEDA, and Y. KIKKAWA. 1976. Morphologic and biochemical study of pulmonary changes induced by bleomycin in mice. Lab. Invest. 35:558-568.Google Scholar
  31. 31.
    WITSCHI, H. P., W. M. HASCHEK, A. J. P. KLIEN-SZANTO, and P. J. HAKKINEN. 1981. Potentiation of diffuse lung damage by oxygen: determining variables. Am. Rev. Respir. Dis. 123:98-103.Google Scholar
  32. 32.
    HASCHEK, W. M. and H. P. WITSCHI. 1979. Pulmonary fibrosis: a possible mechanism. Toxicol. Appl. Pharmacol. 51:475-487.Google Scholar
  33. 33.
    BRIELAND, J. K. and J. C. FANTONE. 1995. Neutrophils and pulmonary fibrosis. In: Pulmonary fibrosis. S. M. Phan, R. S. Thrall, editors. Marcel Dekker, Inc., New York 80:383-397.Google Scholar
  34. 34.
    WEISS, S. J. 1989. Tissue destruction of Neutrophils. New. Engl. J. Med. 320:365-376.Google Scholar
  35. 35.
    KLEBANOFF, S. J. 1988. Phagocytic cells: products of oxygen metabolism. Basic principles and clinical correlates. In: Inflammation. J. I. Gallin, I. M. Goldstein, R. Snyderman, editors. Raven Press, New York. 391-444.Google Scholar
  36. 36.
    KANEKO, M., H. INOUE, R. NAKAZAWA, N. AZUMA, M. SUZUKI, S. YAMAUCHI, and S. B. MARGOLIN. 1988. Pirfenidone induces intercellular adhesion molecule-1 (ICAM-1) down-regulation on cultured human synovial fibroblasts. Clin. Exp. Immunol. 113:72-76.Google Scholar
  37. 37.
    VAN ANDRIAN, V. H., J. D. CHAMBERS, L. M. MCEVOY, R. F. BARGATZE, K. E. ARFORS, and E. C. BUTCHER. 1991. Two-step model of leukocyte-endothelial cell interaction in inflammation: distinct roles for LECAM-1 and the leukocyte β 2 integrins in vivo. Proc. Natl. Acad. Sci. 88:7538-7542.Google Scholar
  38. 38.
    GIRI, S. N., S. L. LEONARD, X. SHI, S. B. MARGOLIN, and V. VALEYATHAN. 1999. Effects of pirfenidone on the generation of reactive oxygen species in vitro. J. Environ. Pathol. Toxicol. Oncol. 18:169-177.Google Scholar
  39. 39.
    CASPARY, W. J., D. A. LANZO, and C. NIZIAK. 1982. Effect of deoxyribonucleic acid on the production of reduced oxygen by bleomycin and iron. Biochemistry 21:334-338.Google Scholar
  40. 40.
    SUIGURA, Y. and T. KIKUCHI. 1978. Formation of superoxide and hydroxy radicals in iron (II)-bleomycin oxygen system: electron spin resonance detection by spin trapping. J. Antibiot. 31:1310-1312.Google Scholar
  41. 41.
    KHALIL, N., C. WHITMAN, Z. LI, D. DANIELPOUR, and A. GREENBERG. 1993. Regulation of alveolar-macrophage TGF-β secretion by corticosteroids in bleomycin-induced pulmonary inflammation in the rat. J. Clin. Invest. 92:1812-1818.Google Scholar
  42. 42.
    KHALIL, N., R. N. O'CONNOR, M. W. UNRUH, P. W. WARREN, K. C. FLANDER, A. KEMP, O. H. BERENZAY, and A. H. GREENBERG. 1991. Increased production and immunohistochemical localization of transforming growth factor-α in idiopathic pulmonary fibrosis. Am. J. Resp. Cell Mol. Biol. 5:155-162.Google Scholar
  43. 43.
    BROCKELMANN, T. J., A. H. LIMPER, T. V. COLBY, and J. A. MCDONALD. 1991. Transforming growth factor β is present at sites of extracellular matrix gene expression in human pulmonary fibrosis. Proc. Natl. Acad. Sci. U.S.A. 88:6642-6646.Google Scholar
  44. 44.
    WAHL, S. M., D. A. HUNT, L. M. WAKEFIELD, N. MCCARTNEY-FRANCIS, L. M. WAHL, and A. B. ROBERTS. 1987. Transforming growth factor β induces monocyte chemotaxis and growth factor production. Proc. Natl. Acad. Sci. U.S.A. 84:5788-5792.Google Scholar
  45. 45.
    POSTLEWAITE, A. E., J. KESKI-OJA, H. L. MOSES, and A. J. KING. 1987. Stimulation of the chemotactic migration of human fibroblasts by transforming growth factor β. J. Exp. Med. 165:251-256.Google Scholar
  46. 46.
    RAGHOW, R., S. LURIE, J. M. SEYER, and A. H. KANG. 1985. Profiles of steady state levels of mRNAs coding for type I procollagen, elastin and fibronectin in hamster lungs undergoing bleomycin-induced interstitial pulmonary fibrosis. J. Clin. Invest. 76:1733-1739.Google Scholar
  47. 47.
    FINE, A. and R. H. GOLDSTEIN. 1987. The effect of TGF-β on cell proliferation and collagen by fibroblasts. J. Biol. Chem. 262:3897-3902.Google Scholar
  48. 48.
    SPORN, M. B., A. B. ROBERTS, L. M. WAKEFIELD, and B. CROMBRUGGHE. 1987. Some recent advances in the chemistry and biology of transforming growth factor β. J. Cell Bio. 105:1039-1045.Google Scholar
  49. 49.
    GIRI, S. N., D. M. HYDE, and M. A. HOLLINGER. 1993. Effect of antibody to TGF-β on bleomycin induced accumulation of lung collagen in mice. Thorax 48:959-966.Google Scholar
  50. 50.
    CHANDLER, D. B., D. M. HYDE, and S. N. GIRI. 1983. Morphometric estimates of cellular changes during the development of bleomycin-induced lung fibrosis in hamsters. Am. J. Pathol. 112:170-177.Google Scholar
  51. 51.
    THRALL, R. S. and R. W. BARTON. 1984. A comparison of lymphocytes population in lung tissue and in bronchoalveolar lavage fluid of rats at various times during the development of bleomycin-induced pulmonary fibrosis. Am. Rev. Respir. Dis. 129:279-283.Google Scholar
  52. 52.
    KRAVIS, T. C., A. AHMED, T. E. BROWN, and J. D. FULMER. 1976. Pathogenic mechanism in pulmonary fibrosis. Collagen-induced migration inhibition factor production and cytotoxicity mediated by lymhocytes. J. Clin. Invest. 58:1223-1232.Google Scholar
  53. 53.
    WESTERMANN, W., R. SCHOBAL, E. B. REIBER, and K. H. FRANK. 1999. Th2 cells as effectors in postirradiation pulmonary damage preceding fibrosis in the rat. Int. J. Radiat. Biol. 5:629-638.Google Scholar

Copyright information

© Plenum Publishing Corporation 2000

Authors and Affiliations

  • Swarnalatha N. Iyer
    • 1
  • Dallas M. Hyde
    • 2
  • Shri N. Giri
    • 1
  1. 1.Department of Molecular BiosciencesPhysiology and Cell Biology School of Veterinary Medicine University of CaliforniaDavis
  2. 2.Department of AnatomyPhysiology and Cell Biology School of Veterinary Medicine University of CaliforniaDavis

Personalised recommendations