Radiation pneumonia, an interstitial pulmonary inflammation, and subsequent radiation lung fibrosis are significant dose-limiting complications and may threaten quality of life for patients receiving radiation to the thorax. Radiation pneumonia and/or pulmonary fibrosis occur in approximately 10-20% of patients treated with thoracic radiotherapy [¹]. The incidence of radiation-induced lung toxicity has increased in recent years due to more aggressive therapies such as combined chemoradiotherapy [²]. Clinical symptoms range from cough, fever, and shortness of breath to death from respiratory failure. At the cellular and tissue level, radiation pneumonia presents as an edema of the interstitial space, infiltration of inflammatory cells, and thickening of the alveolar septa.

Although the molecular mechanism for radiation pneumonia is complex and obscure, involvement of proinflammatory cytokines, chemokines, and cell adhesion molecules has been implicated [³,⁴]. Many investigators have shown that cytokines play essential roles in the pathogenesis of radiation pneumonia [⁵,⁶]. Interleukin-6 (IL-6), which was originally identified as a B-cell differentiation factor [⁷], is now known to be a multifunctional cytokine that regulates acute phase response, immune response, and inflammation [⁸,⁹]. IL-6 is produced by a variety of cells such as T cells, B cells, monocytes, macrophages, fibroblasts, endothelial cells, and several tumor cells [¹⁰]. Clinical as well as experimental findings have suggested the involvement of IL-6 as a pro-inflammatory cytokine in radiation pneumonia [^11-14]. Indeed, up-regulation of IL-6 production has been observed in human and animals with radiation pneumonia.

In this study, we examined the effect of anti-IL-6 monoclonal receptor antibody (IL-6RA) on radiation-induced lung injury in mice having received lethal irradiation of the whole thorax. We investigated whether IL-6RA treatment would offer the promise of a new pharmacologic intervention strategy for ameliorating radiation-induced lung injury.

Radiation pneumonia and subsequent radiation lung fibrosis are major dose-limiting complications for patients undergoing thoracic radiotherapy. Recent research findings support the existence of a mechanism of cellular interaction between lung parenchymal cells and circulating immune cells mediated through various cytokines such as proinflammatory cytokines, chemokines, adhesion molecules, and profibrotic cytokines [³,⁴]. Since IL-6 is a pleiotropic cytokine that plays important roles in the regulation of immune response and inflammation, IL-6 receptor monoclonal antibody treatment has been identified as a promising treatment for Castleman's disease, rheumatoid arthritis, juvenile idiopathic arthritis, and Crohn's disease [^16-19]. IL-6 has also been implicated in the pathogenesis of radiation pneumonia [^11-14] and is synthesized by type II pneumocytes, alveolar macrophages, T lymphocytes, and lung fibroblasts [⁵]. We therefore hypothesized that blockage of the IL-6 signaling pathways may offer an attractive therapeutic target for the amelioration of radiation-induced lung injury.

IL-6RA treatment was found to inhibit a radiation-induced increase in IL-6 proteins 50 and 100 days after irradiation. Moreover, after 150 days IL-6RA significantly suppressed a radiation-induced increase in inflammatory marker SAA proteins in plasma as compared with the control group. Acute phase protein SAA is known as a sensitive systemic marker of inflammation and tissue damage [²⁰]. Further, IL-6 plays an important role in the synergistic induction of the SAA gene and the anti-IL-6 receptor monoclonal antibody inhibits the synergistic induction of SAA [²¹]. These findings indicate that IL-6RA has some beneficial effects. In our study, however, no significant difference was observed in severity of lung damage or length of survival between IL-6RA treated mice and control.

One possible explanation for this finding is that long-term continuous administration of IL-6RA may be necessary for reducing lung toxicity. Rube et al. showed that radiation-induced release of IL-6 in the bronchiolar epithelium of C57BL/6J mice was detected a few hours and several weeks after irradiation (peak at 8 weeks) [¹⁴]. Radiation-induced lung injury is a chronic phenomenon mediated by various cells such as inflammatory cells responding to the release or activation of downstream cytokines, growth factors, or chemokines. Anscher et al. reported long-term (6-month) administration of the small molecule inhibitor of TGF-beta was more effective in reducing radiation-induced lung toxicity than short-term (3-week) administration [²²]. Long-term IL-6RA treatment following irradiation may therefore ameliorate radiation-induced lung injury. Because we were concerned that repeated treatment with rat antibody would result in the production of mouse anti-rat antibodies against the rat antibody, we could not administer IL-6RA more than twice. In a previous pilot study we examined plasma IL-6 levels in mice having received only radiation of 21 Gy and confirmed that radiation-induced IL-6 production in Balb/c mice reached a peak 1 week after irradiation (data not shown). The reason for the choice of this time point was that we wanted to inhibit acute interstitial inflammation.

A marked up-regulation of IL-6 was observed in mice treated with IL-6RA in comparison with the rat IgG group 150 days after irradiation. This result may be explained by the fact that the time until the peak concentration of IL-6 in IL-6RA-treated mice had changed due to the inhibition of autocrine production of IL-6. Rube et al. reported radiation-induced IL-6 production in C57BL/6J mice reached two peaks a few hours and 8 weeks after irradiation [¹⁴]. However, marked mouse strain differences in cytokine levels and patterns may occur such as in the histological configuration of radiation-induced lung injury [²³]. BALB/c mice were chosen for this study because this strain is known to possess comparable radiosensitity [²⁴]. We hypothesized that use of a different mice strain might cause changes in the pathogenesis of radiation pneumonia and thus alter the survival of mice receiving lethally irradiation of the whole thorax. A significant increase in SAA was observed in mice treated with IgG in comparison with the IL-6RA group 150 days after irradiation, whereas the mice treated with IL-6RA showed a significant induction of IL-6 in comparison with the rat IgG group. This result may be explained by the fact that signal transduction of IL-1 or TNF-alpha is more strongly involved in the regulation of SAA production than that of IL-6 [²⁵]. These findings suggest that elevation of IL-6 was not strongly implicated in the pathogenesis of radiation pneumonia but was rather derived from the development of radiation pneumonia. Although a limitation of our study is that a relatively small number of mice were used, it is clear from these results that further intensive study is warranted.

There is concern that intervention to prevent radiation-induced toxicity may also serve to protect cancer because clinical investigations found increased serum IL-6 levels in cancer patients [²⁶]. Aberrant production and signaling of circulating IL-6 has been implicated in tumor generation and poor disease outcome in various cancers [²⁷]. Since blockade of IL-6 signaling by shRNA inhibits lung adenocarinoma cell growth [²⁸], IL-6RA treatment may inhibit radiation-induced lung toxicity as well as tumor proliferation.

To summarize, the findings presented here suggest that early intervention using IL-6RA could not ameliorate radiation-induced lung injury. Additional work is needed to determine the optimal timing and duration for therapy using this approach to the prevention of lung injury after radiation therapy. Despite the negative findings of our study, further intensive studies are needed into strategies for inhibition of cytokine signaling as a way to ameliorating lung toxicity from radiotherapy.

The authors declare that they have no competing interests.

TO carried out the animal experiments and drafted the manuscript. AK and YS performed the animal experiments. TT and IT participated in the statistical analysis. HY, NN, and MN conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.

The authors are indebted to Kumie Hirai, Atsuko Kawaguchi, and Kimiko Sameshima for excellent technical support.