Elsevier

Radiotherapy and Oncology

Volume 72, Issue 3, September 2004, Pages 291-296
Radiotherapy and Oncology

Irradiation leads to susceptibility of hepatocytes to TNF-α mediated apoptosis

https://doi.org/10.1016/j.radonc.2004.07.001Get rights and content

Abstract

Background and purpose

The pathogenesis of radiation-induced-liver-disease (RILD) is still unknown. We tested the hypothesis that irradiated liver macrophages influence the viability of radiation stressed hepatocytes.

Patients and methods

Hepatocytes and liver macrophages were isolated from rat liver, cultured and irradiated with doses of 2, 8, and 25 Gy. Cell viability was measured by trypan blue exclusion, and by annexin V/propidium iodide staining. TNF-α in the supernatants from liver macrophage cell culture was quantitatively detected by ELISA. TNF-α mRNA from liver macrophages was measured by real time PCR.

Results

Irradiation had no influence on cell viability. Apoptosis of irradiated hepatocytes was detected 24 h after replacing 50% of medium with supernatants of irradiated liver macrophages 6 h after irradiation (32.0±5.8% compared to solely irradiated cells (12±2.9%, P=0.02)). In supernatants of hepatocytes, no TNF-α secretion could be measured. A radiation dependent increase was found in supernatants of liver macrophages. Addition of anti-TNF-α-antibodies to the supernatants of irradiated liver macrophages reduced apoptosis (20±0.9%). Incubation of irradiated hepatocytes with purified recombinant TNF-α increased apoptosis in irradiated hepatocytes. This effect could be abrogated by additional administration of TNF-α-antibodies.

Conclusions

Irradiation leads to susceptibility of hepatocytes to TNF-α mediated apoptosis. Liver macrophages may be one of the sources of TNF-α in case of liver-irradiation. This cell–cell-interaction may be an important initial step towards RILD and liver fibrosis.

Introduction

Although hepatocytes are considered more radioresistant than other cells, the liver is a highly radiosensitive organ [1], [2], [3], [17], [18], [19], [20]. Therefore, radiation-induced liver disease (RILD) is a dose limiting complication in abdominal irradiation. This plays a decisive role for radioimmunotherapy of malignant lymphomas, radio-chemotherapy in biliopancreatic carcinomas, and stereotactic radiotherapy of liver metastases [35]. The threshold dose for whole liver irradiation is supposed to be between 20 and 30 Gy [16]. A large volume effect for RILD has been shown so that the mean liver dose plays an important role [5], [7].

The acute clinical period of RILD after irradiation tends to be relatively silent, the subacute phase is characterized by the development of anicteric ascites, elevation of liver enzymes, rapid weight gain, and liver enlargement 2 weeks to 4 months after treatment [23].

In animal studies an initial inflammatory reaction after irradiation is not followed by a recovery phase and complete restitution. Instead, a progressive liver fibrosis and cirrhosis is regularly found [11].

The molecular pathogenesis of hepatocellular death after irradiation and development of irreversible RILD is still obscure. Cytokines are important for hepatocellular damage, repair and fibrosis development in other toxic liver injuries [15], [28], [29]. Different cell types interact via cytokines in the development of normal tissue reactions after radiotherapy [12]. Therefore, the molecular mechanisms of RILD might be similar. The damage induced by irradiation may lead to development of liver fibrosis and may not be followed by restitutio ad integrum in contrast to toxic injuries.

In the present work, we studied the influence of radiation on viability of hepatocytes in culture. Secondly, we tested the hypothesis that irradiated liver macrophages influence the viability of radiation stressed hepatocytes.

Section snippets

Cell isolation and culture conditions

Hepatocytes were isolated from normal animals (wistar rats, Harlan Winkelmann, Borchen, Germany) according to Seglen [33] as described previously [30]. The treatment of the rats was approved by the local committee on animal welfare. Purity of cell isolations was determined by phase-contrast microscopy, and by immunocytochemistry using antibodies against laminin or GFAP of stellate cells (both Sigma, Deisenhofen, Germany) or ED1 and ED2 (gift from C. Dijkstra, [9]) for macrophages [27].

The cells

Viability of hepatocytes after irradiation

Twenty four and 48 h after irradiation of primary rat hepatocytes, living cells were determined by trypan blue exclusion (Table 1). After 2 Gy irradiation, no significant difference in cell survival 24 h (P=0.55) and 48 h (P=0.1) post irradiation could be detected. In the same way, irradiation with 8 Gy had no influence on cell survival (P=0.33 and 0.07). Even irradiation with 25 Gy did not decrease the viability of hepatocytes 24 h (P=0.51) and 48 h (P=0.27) after irradiation.

Apoptosis of irradiated hepatocytes after incubation with cell culture supernatants from irradiated liver macrophages

No radiation-induced

Discussion

In this work, we demonstrate that irradiation of cultured rat hepatocytes does not lead to cytotoxicity. Irradiation of liver macrophages induces TNF-α release into the culture medium. Treatment of irradiated hepatocytes with supernatants from irradiated liver macrophages significantly increases apoptosis. This effect can be observed up to 12 h after irradiation. Twenty-four hours after irradiation, hepatocytes have recovered their full anti-apoptotic potential. The data show that hepatocytes

Acknowledgements

This work was supported by a grant of DFG-SFB 402TPC6,D3 and of the Medical Faculty of the University of Goettingen.

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    Hans Christiansen and Bernhard Saile contributed equally to this work.

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