Long-term alterations of cytokines and growth factors expression in irradiated tissues and relation with histological severity scoring

Abstract

Purpose: Beside its efficacy in cancer treatment, radiotherapy induces degeneration of healthy tissues within the irradiated area. The aim of this study was to analyze the variations of proinflammatory (IL-1α, IL-2, IL-6, TNF-α, IFN-γ), profibrotic (TGF-β1), proangiogneic (VEGF) and stem cell mobilizing (GM-CSF) cytokines and growth factors in an animal model of radiation-induced tissue degeneration.

Materials and methods: 24 rats were irradiated unilaterally on the hindlimb at a monodose of 30 Gy. Six weeks (n=8), 6 months (n=8) and 1 year (n=8) after irradiation the mediators expression in skin and muscle were analyzed using Western blot and the Bio-Plex® protein array (BPA) technology. Additional histological severity for fibrosis, inflammation, vascularity and cellularity alterations scoring was defined from histology and immnunohistochemistry analyses.

Results: A significant increase of histological severity scoring was found in irradiated tissue. Skin tissues were more radio-sensitive than muscle. A high level of TGF-β1 expression was found throughout the study and a significant relation was evidenced between TGF-β1 expression and fibrosis scoring. Irradiated tissue showed a chronic inflammation (IL-2 and TNF-α significantly increased). Moreover a persistent expression of GM-CSF and VEGF was found in all irradiated tissues. The vascular score was related to TGF-β1 expression and the cellular alterations score was significantly related with the level of IL-2, VEGF and GM-CSF.

Conclusion: The results achieved in the present study underline the complexity and multiplicity of radio-induced alterations of cytokine network. It offers many perspectives of development, for the comprehension of the mechanisms of late injuries or for the histological and molecular evaluation of the mode of action and the efficacy of rehabilitation techniques.

Figure 1. Simplified model of the complex network of interacting processes and signals in the pathogenesis of radio-induced injury.

The main mediators of the four cytokine and growth factor families involved in inter-cellular exchanges during radiation-induced tissue degeneration in skin and muscles are.represented, namely the expression of proinflammatory (IL-1α, IL-2, IL-6, TNF-α, IFN-γ), profibrotic (TGF-β1), proangiogenic (VEGF) and stem cell mobilizing (GM-CSF) mediators.

Figure 2. Scale of severity for inflammation, fibrosis, vascularity and cellular alterations.

The inflammation score, evaluated on hematoxylin eosin-stained slides, was based on the presence of inflammatory infiltrates. The fibrosis score evaluated on sirius red-stained slides, depend on the intensity of the fibrosis and the modification of the tissular structure. The vascular score was evaluated both on sirius red- and FVIII-stained slides. It was calculated from the number and the alterations of structure of vessels. The cellular alterations score was evaluated on hematoxylin eosin-stained slides. In skin, it was calculated according to the loss and necrosis of cells as well as the number of myofibroblasts. In muscles, it depends mainly on the presence of dystrophic nuclei and necrosis.

Figure 3. Evolution of the score of fibrosis, inflammation, vascularity and cellular alterations with time in skin and muscle.

The fibrosis score showed an increased with time in both skin and muscle. In skin, the inflammation score appeared to decrease at 6 months, but increased at 1 year. In muscle, the inflammation score increased consistently with time.

Figure 4. Mean differences of expression of different mediators between healthy skin and irritated skin over time.

All pro-inflammatory mediators (IL-1α, TNF-α, IL-2, IFN-γ, IL-6), the pro-angiogenic mediator VEGF, and the stem cell-mobilizing cytokine GM-CSF evolved consistently with time, the level of expression measured at 6 weeks appeared to decrease at 6 months, then the trend reversed at 1 year. The expression of the profibrotic mediator TGF-β remained markedly increased during the study. (*: p<0.05 and **: p<0.01)

Figure 5. Variation of expression of cytokine and growth factor for each score of severity from inflammation, fibrosis, vascularity and cellular alterations in skin and muscles.

In skin only significant variations are shown. The y-axis corresponds to the level of expression of each cytokine in ng/µg of total protein.