Benotmane, R.

Dettmering, T.

Durante, M.

Erbeldinger, N.

Fournier, C.

Lowe, D.

Raj, K.

Tapio, S.

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Inflammatory cytokine release by vascular cells after X-irradiation∗T. Dettmering1, N. Erbeldinger1, D. Lowe2, M. Durante1, R. Benotmane3, S. Tapio4, K. Raj2, andC. Fournier11GSI, Darmstadt, Germany; 2Public Health England, Oxford, UK; 3SCK-CEN, Belgium; 4HelmholtzZentrumMu¨nchen, GermanyIntroductionExposure to high radiation doses is considered to be re-sponsible for the development of cardiovascular diseases(CVD), but also low doses are suspected to be a risk factor[1,2]. The development of CVD is mediated by changesin the cytokine release of endothelial and immune cells,whereas the role of smooth muscle cells is underinvesti-gated up to now [3]. We used a co-culture model of bothcell types in order determine the cytokine release up to twoweeks after irradiation, comparing low and high doses ofX-rays. Here we present the results of Interleukin-6 (IL-6),a cytokine which can have both pro- and anti-inflammatoryeffects.Materials and MethodsHuman coronary artery endothelial cells (HCAEC) andhuman smooth muscle cells (SMC) were immortalized byhTERT overexpression. Cell types were either culturedseparately or in co-culture for 3 d prior irradiation. Af-ter irradiation with 0.05, 0.1, 0.5 and 2 Gy X-rays, mediumsupernatants were collected after 4 h, 24 h, 1 week and 2weeks and frozen at −80 ◦C. Medium was replaced 24 hbefore each time point. Concentrations of 21 inflammation-related cytokines in the supernatants of mono- and co-cultures were determined by bead-based protein arrays.Based on these results, IL-6 concentrations were quantifiedusing ELISA (eBioscience) and data were analyzed usinga custom pipeline written in the R language.Results and DiscussionIn Figure 1, the IL-6 release of unirradiated cells isshown over the time. While the IL-6 release rate ofHCAEC remained almost unchanged, SMC and the cocul-ture released initially high amounts of IL-6 that decreasedrapidly during the following week. The IL-6 productionof the co-culture after assembly exceedes the total amountof the two monocultures, pointing towards signaling pro-cesses occuring in the co-cultured cells.There was no significant radiation-induced change of IL-6 release 4 h, 24 h or 2 weeks after radiation exposure (notshown). In contrast, 1 week after irradiation the IL-6 con-centrations were 1.5× (0.5 Gy) and 2× (2 Gy) increasedrelative to control (Fig. 2). So far, this indicates no influ-ence of very low doses (≤ 0.1 Gy) on the release of this∗The research leading to these results has received funding fromthe Euratom Seventh Framework Programme under grant agreement no.295823 (“PROCARDIO”).inflammatory mediator. Higher doses (≥ 0.5 Gy), how-ever, are able to increase the cytokine release in a vascu-lar co-culture model, but mono-cultures remain unaffectedby irradiation. Preliminary results from proteomics stud-ies showed an induction of proteins that could be indicativeof ongoing inflammation, thus supporting our results (notshown). Preliminary transcriptomic data of HCAEC showthat genes involved in regulation of mitosis were stronglydownregulated at 24 h post-IR, pointing towards a G2/Marrest (not shown). After 7 days, these genes are slightlybut significantly upregulated after 2 Gy irradiation.01000200030004h 1d 7d 14dTime after irradiationIL−8 (pg/ml/105cells/24 h) CelltypeHCAECSMCCocultureFigure 1: IL-6 cytokine release rate of unirradiated cells asa function of the time. Each sample was measured at leasttwice. Error bars show SEM. N = 2, n = 6HCAEC SMC Coculture*******12340 0.05 0.1 0.5 2 0 0.05 0.1 0.5 2 0 0.05 0.1 0.5 2Dose (Gy)IL−6 concentration relative to 0 GyFigure 2: IL-6 cytokine release one week post irradiation.Concentrations have been normalized to 0 Gy sample. Er-ror bars show SEM. *** p < 0.001; **** p < 0.0001.N = 3, n ≥ 9References[1] Boerma and Hauer-Jensen. Cardiol Res Pract. 2010 Oct 4;2011. pii: 858262.[2] Adams et al. Semin Radiat Oncol. 2003 Jul; 13(3):346–56.[3] Schultz-Hector et al. Int J Radiat Oncol Biol Phys 2007 Jan1; 67(1):10–8.GSI SCIENTIFIC REPORT 2013 BIOPHYSICS-11doi:10.15120/GR-2014-1-BIOPHYSICS-11 213

Inflammatory cytokine release by vascular cells after X-irradiation

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Inflammatory cytokine release by vascular cells after X-irradiation

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