Modulation of radiation-induced tumour necrosis factor α (TNF-α) expression in the lung tissue by pentoxifylline

Abstract

Purpose: The lung is the major dose-limiting organ for radiotherapy of cancer in the thoracic region. Immediate cellular damage after irradiation is supposed to result in cytokine-mediated multicellular interactions with induction and progression of inflammatory and fibrotic tissue reactions. Pentoxifylline (PTX) down-regulates the production of proinflammatory cytokines, particularly TNF-α, in response to noxious stimuli and may therefore provide protection against radiation-induced, cytokine-mediated cellular damage. The purpose of this study was to investigate the temporal and spatial release of TNF-α in the lung tissue after thoracic irradiation with 12 Gy. In addition, we evaluated the ability of PTX to reduce the radiation-induced TNF-α release in this animal model of thoracic irradiation.

Materials and methods: C57BL/6J mice were exposed to either sham irradiation or single fraction of 12 Gy delivered to the thorax. Four study groups were defined: those that received neither irradiation nor PTX (NT group), those that received PTX but no irradiation (PTX group), those that underwent irradiation without PTX (XRT group) and those that received both PTX and irradiation (PTX/XRT group). Treated and sham-irradiated mice were sacrificed corresponding to the latent period and the pneumonic phase. The TNF-α mRNA expression in the lung tissue was quantified by ‘real-time’ quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Immunohistochemical detection methods (alkaline phosphatase anti-alkaline phosphatase (APAAP)) and automated image analysis were used for objective quantification of TNF-α protein expression.

Results: Following thoracic irradiation with a single dose of 12 Gy (XRT group), radiation-induced TNF-α mRNA release in the lung tissue was significantly increased during the acute phase of pneumonitis (P<0.05). The elevated levels of TNF-α mRNA during the pneumonic phase correlate with a significant increase of positive inflammatory cells, predominantly macrophages, in the lung parenchyma (P<0.05). In contrast to the radiation-only group (XRT-group), the lung tissue of the PTX-treated mice (PTX/XRT group) revealed only a minor radiation-mediated TNF-α response on mRNA and protein level.

Conclusions: This study demonstrates a significant radiation-induced increase of TNF-α (on mRNA and protein level) in the lung tissue during the pneumonic phase. The predominant localisation of TNF-α in areas of inflammatory cell infiltrates suggests involvement of this cytokine in the pathogenesis of radiation-induced lung injury. In addition, we observed a pronounced reduction of the TNF-α mRNA and protein production in the study group that received both PTX and radiation (PTX/XRT group) as compared to the radiation-only group (XRT group). Therefore our results indicate that PTX down-regulates the TNF-α mRNA and protein production in the lung tissue in response to radiation.

Introduction

The radiosensitivity of the lung tissue limits the dose of radiation which can be delivered to tumours in the thoracic region. Radiation-induced lung damage may arise depending on the total dose of radiation, the fractionation schedule, the volume of lung tissue irradiated, the existence of prior lung disease and the use of chemotherapeutic drugs in the treatment of the disease [30], [32], [40]. Damage to endothelial or epithelial cells is assumed to be the initial step leading to radiation pneumonitis and ultimately to pulmonary fibrosis. But the process of injury and repair initiated by irradiation is also a function of activation of cells to produce important biological mediators, such as cytokines, which modulate diverse aspects of the inflammatory and fibrogenic response. Therefore, the pathophysiological tissue response after lung irradiation implies the induction of numerous cytokines which form the basis for the multicellular interactions of the inflammatory and fibrogenic process associated with radiation injury [11], [12], [21], [22], [34], [35], [42]. The relative role of cytokine dysregulation versus direct tissue injury from irradiation for the pathogenesis of radiation pneumonitis/fibrosis remains elusive.

TNF-α is thought to be a key mediator for the pathogenesis of radiation pneumonitis, because it shows the following spectrum of biological activities. TNF-α exerts in particular proinflammatory effects by inducing the expression of adhesion molecules that recruit leukocytes into the sites of tissue damage, by priming leukocytes for oxidant production, and by inducing production of prostaglandins and other mediators of inflammation. TNF-α inhibits anti-coagulatory mechanisms and therefore promotes thrombotic processes [29]. In addition, TNF-α exerts fibrogenic effects by stimulating the growth of fibroblasts and increasing the collagen deposition. [19]. Therefore, a pharmacological regulation of the TNF-α production at the initial stage could possibly halt the progression of radiation-induced injury. A drug which suppresses the production of TNF-α but lacks the many side effects of glucocorticoids might be useful as an anti-inflammatory agent.

Pentoxifylline (PTX) is a xanthine derivate that has generated widespread interest in the field of oncology based on its reported potential ability to ameliorate radiation- and chemotherapy-induced toxicity. PTX has been shown to enhance microvascular blood flow and decrease platelet aggregation, thereby maintaining perfusion in radiated tissues. In addition, PTX down-regulates the production of proinflammatory cytokines, particularly TNF-α, in response to noxious stimuli and inhibits granulocyte-mediated cytotoxicity after TNF-α exposure and may, therefore, provide protection against radiation-induced, cytokine-mediated cellular damage [2], [8], [38], [44], [45]. Since PTX has long been used in the treatment of peripheral vascular disease, the drug is widely available and low toxicity has been demonstrated.

The purpose of this study was to investigate the temporal and spatial release of TNF-α in the lung tissue after thoracic irradiation with 12 Gy. In addition, we evaluated the ability of PTX to reduce the radiation-induced TNF-α release and the lung toxicity in this animal model of thoracic irradiation.