Dose-dependent induction of transforming growth factor beta (TGF-beta) in the lung tissue of fibrosis-prone mice after thoracic irradiation

Purpose: The lung is the major dose-limiting organ for radiotherapy of canc er in the thoracic region. The pathogenesis of radiation-induced lung injur y at the molecular level is still unclear. Immediate cellular damage after irradiation is supposed to result in cytokine-mediated multicellular intera ctions with induction and progression of fibrotic tissue reactions. The pur pose 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 and Materials: The thoraces of C57BL/6 mice were irradiated with 6 and 12 Gy, respectively. Treated and sham-irradiated control mice mere sacr ificed 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 we eks postirradiation), and the beginning of the fibrotic phase (2 1 weeks po stirradiation), The lung tissue from three different mice per dosage and ti me 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 react ion (RT-PCR); the cellular origin of the TGF-beta protein was identified by immunohistochemical staining (alkaline phosphatase-anti-alkaline phosphata se [APAAP]). The cytokine expression on mRNA and protein level was correlat ed with the histopathological alterations. Results: Following thoracic irradiation with a single dose of 12 Gy, radiat ion-induced TGF-beta release in lung tissue was appreciable already within the first hours (1, 3, and 6 hours postirradiation) and reached a significa nt increase after 12 hours; subsequently (48 hours, 72 hours, and 1 week po stirradiation) the TGF-beta expression declined to basal levels. At the beg inning of the pneumonic phase, irradiation-mediated stimulation of TGF-beta release reached maximal values at 2 and 4 weeks. The elevated levels of TG F-beta mRNA during the latent phase have been found to correlate with immun ohistochemical staining of alveolar macrophages. The most striking increase in TGF-beta immunoreactivity was seen during the acute phase of pneumoniti s. Throughout this observation period, type II pneumocytes and fibroblasts (apart from inflammatory cells) served as important sources of TGF-beta exp ression. Increased TGF-beta expression was detected prominently in regions of histopathologic radiation injury. After exposure to a single radiation d ose of 6 Gy, the lung tissue revealed only a minor radiation-mediated TGF-b eta 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 d uring the later time points revealed no significant alterations in comparis on 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 wi th 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 shoul d be performed to explore the role of other cytokines in the development of radiation injury. An improved understanding of the underlying mechanisms o f pulmonary fibrosis may eventually lead to modulatory intervention at the molecular level to modify the fibrotic process. (C) 2000 Elsevier Science I nc.