Thoracic Irradiation Recruit M2 Macrophage into the Lung, Leading to Pneumonitis and Pulmonary Fibrosis

  • Park, Hae-Ran (Radiation Division for Biotechnology, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute) ;
  • Jo, Sung-Kee (Radiation Division for Biotechnology, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute) ;
  • Jung, Uhee (Radiation Division for Biotechnology, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute)
  • Received : 2017.08.22
  • Accepted : 2017.11.03
  • Published : 2017.12.31


Background: Radiation-induced pneumonitis and pulmonary fibrosis are common dose-limiting complications in patients receiving radiotherapy for lung, breast, and lymphoid cancers. In this study, we investigated the characteristics of effective immune cells related to pneumonitis and fibrosis after irradiation. Materials and Methods: After anesthesia, the whole thorax of C57BL/6 mice was irradiated at 14 Gy. The lung tissue and bronchoalveolar lavage fluid were collected at defined time points post-irradiation for the determination of histological and immunohistochemical analysis and inflammatory cell population infiltrated into the lung. Results and Discussion: Whole thoracic irradiation increased the deposition of extracellular matrix (ECM), lung weight, and pleural effusions, which started to die from 4 months later. At 4 months after irradiation, the numbers of macrophages and lymphocytes as well as neutrophils were increased dramatically in the lung. Interestingly, the macrophages that were recruited into the lung after irradiation had an enlarged foamy morphology. In addition, the expressions of chemokines (CCL-2, CCL-3, CXCL-10) for the attraction of macrophages and T cells were higher in the lung of irradiated mice. The high expressions of these chemokines were sustained up to 6 months following irradiation. In thoracic irradiated mice, infiltrated macrophages into the lung had the high levels of Mac-3 antigens on their surface and upregulated the hallmarks of alternatively activated macrophages such as arginase-1 and CD206. Furthermore, the levels of IL-4 and IL-13 were higher in a BAL fluid of irradiated mice. Conclusion: All results show that thoracic irradiation induces to infiltrate various inflammation-related immune cells, especially alternatively activated macrophages, through enhancing the expression of chemokines, suggesting that alternatively activated macrophages are most likely important for leading to pulmonary fibrosis.


Supported by : Korea Atomic Energy Research Institute (KAERI)


  1. Mehta V. Radiation pneumonitis and pulmonary fibrosis in nonsmall- cell lung cancer: pulmonary function, prediction, and prevention. Int. J. Radiat. Oncol. Biol. Phys. 2005;63:5-24.
  2. Thannickal VJ, Toews GB, White ES, Lynch III JP, Martinez FJ. Mechanisms of pulmonary fibrosis. Ann. Rev. Med. 2004;55:395-417.
  3. Wynn TA. Integrating mechanisms of pulmonary fibrosis. J. Exp. Med. 2011;208:1339-1350.
  4. Wilson MS, Wynn TA. Pulmonary fibrosis: pathogenesis, etiology and regulation. Mucosal Immunol. 2009;2:103-121.
  5. Wynn TA. Cellular and molecular mechanisms of fibrosis. J. Pathol. 2008;214:199-210.
  6. Wynn TA, Ramalingam TR. Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nat. Med. 2012;18:1028-1040.
  7. Duffield JS, et al. Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair. J. Clin. Invest. 2005;115:56-65.
  8. Johnston CJ, Wright TW, Rubin P, Finkelstein JN. Alterations in the expression of chemokine mRNA levels in fibrosis-resistant and -sensitive mice after thoracic irradiation. Exp. Lung Res. 1998;24:321-337
  9. Johnston CJ, Williams JP, Okunieff P, Finkelstein JN. Radiationinduced pulmonary fibrosis: examination of chemokines and chemokines receptor families. Radiat. Res. 2002;157:256-265.[0256:RIPFEO]2.0.CO;2
  10. Park HR, Jo SK, Yu DK, Jung UH. Fractionated irradiations lead to chronic allergic airway inflammation through increasing the influx of macrophages. Inflamm. Res. 2013;62:27-36.
  11. Buttner C, et al. Local production of interleukin-4 during radiation- induced pneumonitis and pulmonary fibrosis in rats: macrophages as a prominent source of interleukin-4. Am. J. Respir. Cell Mol. Biol. 1997;17:315-325.
  12. Chiang CS, et al. Compartmental responses after thoracic irradiation of mice: strain differences. Int. J. Radiat. Oncol. Biol. Phys. 2005;62:862-871.
  13. Yarnold J, Brontons MCV. Pathogenetic mechanisms in radiation fibrosis. Radiother. Oncol. 2010;97:149-161.
  14. Zhang H, et al. The development of classically and alternatively activated macrophages has different effects on the varied stages of radiation-induced pulmonary injury in mice. J. Radiat. Res. 2011;52:717-726.
  15. Zaidi A, Jelveh S, Mahmood J, Hill RP. Effects of lipopolysaccharide on the response of C57BL/6J mice to whole thorax irradiation. Radiother. Oncol. 2012;105:341-349.
  16. Gordon S, Taylor PR. Monocyte and macrophage heterogeneity. Nat. Rev. Immunol. 2005;5:953-964.
  17. Murray PJ, Wynn TA. Protective and pathogenic functions of macrophage subsets. Nat. Rev. Immunol. 2011;11:723-737.
  18. Gordon S, Martinez FO. Alternative activation of macrophages: mechanism and functions. Immunity. 2010;32:593-604.
  19. Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M. The chemokines system in diverse forms of macrophage activation and polarization. Trends Immunol. 2004;12:677-686.
  20. Nair MG, Cochrane DW, Allen JE. Marophages in chronic type 2 inflammation have a novel phenotype characterized by the abundant expression of Ym1 and Fizz1 that can be partly replicated in vitro. Immunol. letters. 2003;85:173-180.
  21. Jackson IL, Vujaskovic Z, Down JD. Revisiting strain-related differences in radiation sensitivity of the mouse lung: recognizing and avoiding the confounding effects of pleural effusions. Radiat. Res. 2010;173:10-20.
  22. Dileto CL, Travis EL. Fibroblast radiosensitivity in vitro and lung fibrosis in vivo: comparison between a fibrosis-prone and fibrosis- resistant mouse strain. Radiat. Res. 1996;146:61-67.
  23. Franko AJ, Sharplin J, Chahary A, Barcellos-Hoff MH. Immunohistochemical localization of transforming growth factor ${\beta}$ and tumor necrosis factor ${\alpha}$ in the lungs of fibrosis-prone and "nonfibrosing" mice during the latent period and early phase after irradiation. Radiat. Res. 1997;147:245-256.
  24. Johnston CJ, Piedboeuf B, Baggs R, Rubin P, Finkelstein JN. Differences in correlation of mRNA gene expression in mice sensitive and resistant to radiation-induced pulmonary fibrosis. Radiat. Res. 1995;142:197-203
  25. Johnston CJ, Wiliams JP, Elder A, Hernady E, Finkelstein JN. Inflammatory cell recruitment following thoracic irradiation. Exp. Lung Res. 2004;30:369-382.
  26. Rubin P, Finkelstein JN, Shapiro D. Molecular biology mechanisms in the radiation induction of pulmonary injury syndromes: interrelationship between the alveolar macrophage and the septal fibroblast. Int. J. Radiat. Oncol. Biol. Phy. 1992;24: 93-101.
  27. Gabbiani G. The myofibroblasts in wound healing and fibrocontractive diseases. J. Pathol. 2003;200:500-503.
  28. Dougherty GJ, McBride WH. Macrophage heterogeneity. J. Clin. Lab. Immunol. 1984;14:1-11.
  29. Springer TA. Monoclonal antibody analysis of complex biological systems. Combination of cell hybridization and immunoadsorbents in a novel cascade procedure and its application to the macrophage cell surface. J. Biol. Chem. 1981;256:3833-3839.
  30. Ho MK, Springer TA. Tissue distribution, structural characterization, and biosynthesis of Mac-3, a macrophage surface glycoprotein exhibiting molecular weight heterogeneity. J. Biol. Chem. 1983;258:636-642.
  31. Sun L, et al. New concepts of IL-10-induced lung fibrosis: fibrocytes recruitment and M2 activation in a CCL2/CCR2 axis. Am. J. Physiol. Lung Cell Mol. Physiol. 2011;300:L341-L353.
  32. Varin A, Goron S. Alternative activation of macrophages: immune function and cellular biology. Immunobiology. 2009;214: 630-641.
  33. Mosser DM, Edwards JP. Exploring the full spectrum of macrophage activation. Nat. Rev. Immunol. 2008;8:958-969.
  34. Anthony RM, et al. Memory T(H)2 cells induce alternatively activated macrophages to mediate protection against nematode parasites. Nat. Med. 2006;12:955-960.
  35. Park HR, Jo SK, Paik SG. Factors effecting the Th2-like immune response after gamma-irradiation: low production of IL-12 heterodimer in antigen-presenting cells and small expression of the IL-12 receptor in T cells. Int. J. Radiat. Biol. 2005;81:221-231.
  36. Park HR, Jo SK, Eom HS. Chronic effects of single and fractionated ${\gamma}$-irradiation on an impairment of Th1-related immune response. Int. J. Radiat. Biol. 2011;87:534-543.
  37. Song E, Ouyang N, Horbelt M, Antus B, Wang M, Exton MS. Influence of alternatively and classically activated macrophages on fibrogenic activities of human fibroblasts. Cell. Immunol. 2000;204:19-28.
  38. Chiaramonte MG, Donaldson DD, Cheever AW, Wynn TA. An IL-13 inhibitor blocks the development of hepatic fibrosis during a T-helper type 2-dominated inflammatory response. J. Clin. Invest. 1999;104:777-785.
  39. Huaux F, Liu T, McGarry B, Ullenbruch M, Phan SH. Dual roles of IL-4 in lung injury and fibrosis. J. Immunol. 2003;170:2083-2092.
  40. Kolodsick JE, et al. Protection from fluorescein isothiocyanateinduced fibrosis in IL-13-deficient, but not IL-4-deficient, mice results from impaired collagen synthesis by fibroblasts. J. Immunol. 2004;172:4068-4076.
  41. Yang G, et al. Anti-IL-13 monoclonal antibody inhibits airway hyperresponsiveness, inflammation and airway remodeling. Cytokine 2004; 28:224-232.
  42. Lumsden RV, et al. Modulation of pulmonary fibrosis by IL- 13R${\alpha}$2. Am. J. Physiol. Lung Cell Mol. Physiol. 2015;308:L710-L718.
  43. Su S, Zhao Q, et al. miR-142-5p and miR-130a-3p are regulated by IL-4 and IL-13 and control profibrogenic macrophage program. Nat. Commun. 2015;6:8523.
  44. Groves AM, Johnston CJ, Misra RS, Williams JP, Finkelstein JN. Effects of IL-4 on pulmonary fibrosis and the accumulation and phenotype of macrophage subpopulations following thoracic irradiation. Int. J. Radiat. Biol. 2016;92:754-765.
  45. Giri SN, Hyde DM, Marafino BJ Jr. Ameliorating effect of murine interferon-${\gamma}$ on bleomycin-induced lung collagen fibrosis in mice. Biochem. Med. Meta. Biol. 1986;36:194-197.
  46. Gurujeyalakshmi G, Giri SN. Molecular mechanisms of antifibrotic effect of interferon-${\gamma}$ in bleomycin mouse model of lung fibrosis: downregulation of TGF-${\beta}$ and procollagen I and III gene expression. Exp. Lung Res. 1995;21:791-808.
  47. Ulloa L, Doody J, Massague J. Inhibition of transforming growth factor-${\beta}$/SMAD signaling by the interferon-${\gamma}$/STAT pathway. Nature. 1999;397:710-713.