Dose-dependent induction of transforming growth factor beta (TGF-beta) in the lung tissue of fibrosis-prone mice after thoracic irradiation.
OBJECTIVE
The lung is the major dose-limiting organ for radiotherapy of cancer in the thoracic region. The pathogenesis of radiation-induced lung injury at the molecular level is still unclear. Immediate cellular damage after irradiation is supposed to result in cytokine-mediated multicellular interactions with induction and progression of fibrotic tissue reactions. The purpose of this investigation was to evaluate the acute and long-term effects of radiation on the gene expression of transforming growth factor beta (TGF-beta) in a model of lung injury using fibrosis-sensitive C57BL/6 mice.


METHODS
The thoraces of C57BL/6 mice were irradiated with 6 and 12 Gy, respectively. Treated and sham-irradiated control mice were sacrificed at times corresponding to the latent period (1, 3, 6, 12, 24, 48, 72 hours and 1 week postirradiation), the pneumonic phase (2, 4, 8, and 16 weeks postirradiation), and the beginning of the fibrotic phase (24 weeks postirradiation). The lung tissue from three different mice per dosage and time point was analyzed by a combination of polymerase chain reaction (PCR), immunohistochemistry, and light microscopy. The mRNA expression of TGF-beta was quantified by competitive reverse transcriptase/polymerase chain reaction (RT-PCR); the cellular origin of the TGF-beta protein was identified by immunohistochemical staining (alkaline phosphatase-anti-alkaline phosphatase [APAAP]). The cytokine expression on mRNA and protein level was correlated with the histopathological alterations.


RESULTS
Following thoracic irradiation with a single dose of 12 Gy, radiation-induced TGF-beta release in lung tissue was appreciable already within the first hours (1, 3, and 6 hours postirradiation) and reached a significant increase after 12 hours; subsequently (48 hours, 72 hours, and 1 week postirradiation) the TGF-beta expression declined to basal levels. At the beginning of the pneumonic phase, irradiation-mediated stimulation of TGF-beta release reached maximal values at 2 and 4 weeks. The elevated levels of TGF-beta mRNA during the latent phase have been found to correlate with immunohistochemical staining of alveolar macrophages. The most striking increase in TGF-beta immunoreactivity was seen during the acute phase of pneumonitis. Throughout this observation period, type II pneumocytes and fibroblasts (apart from inflammatory cells) served as important sources of TGF-beta expression. Increased TGF-beta expression was detected prominently in regions of histopathologic radiation injury. After exposure to a single radiation dose of 6 Gy, the lung tissue revealed only a minor radiation-mediated TGF-beta mRNA response. The modest upregulation ranged from 6 hours to 48 hours after irradiation. Corresponding to the only minor histopathologic changes after thoracic irradiation with 6 Gy, measurement of TGF-beta mRNA levels during the later time points revealed no significant alterations in comparison to untreated control mice.


CONCLUSIONS
This study demonstrates an acute and long-lasting increase in the expression of TGF-beta in lung tissue following thoracic irradiation with 12 Gy. The predominant localization of TGF-beta in areas of inflammatory cell infiltrates and fibrosis suggests involvement of this cytokine in the pathogenesis of radiation-induced pulmonal fibrosis. Further studies should be performed to explore the role of other cytokines in the development of radiation injury. An improved understanding of the underlying mechanisms of pulmonary fibrosis may eventually lead to modulatory intervention at the molecular level to modify the fibrotic process.
Rube
C E
CE
Department of Radiooncology, University of Munster, Munster, Germany. ruebe@med-rz.uni-saarland.de
Uthe
D
D
Schmid
K W
KW
Richter
K D
KD
Wessel
J
J
Schuck
A
A
Willich
N
N
Rube
C
C
D000818:Animals; D004307:Dose-Response Relationship, Radiation; D005260:Female; D015870:Gene Expression / Q000528:radiation effects; D008168:Lung / Q000378:metabolism / Q000473:pathology / Q000528:radiation effects*; D051379:Mice; D008810:Mice, Inbred C57BL; D016133:Polymerase Chain Reaction; D011658:Pulmonary Fibrosis / Q000209:etiology / Q000378:metabolism* / Q000473:pathology; D012333:RNA, Messenger / Q000378:metabolism; D017564:Radiation Pneumonitis / Q000209:etiology / Q000378:metabolism* / Q000473:pathology; D011853:Radiobiology; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000378:metabolism / Q000528:radiation effects*
D012333:RNA, Messenger; D016212:Transforming Growth Factor beta