Durante, M.

Fournier, C.

Madl, L.

Podda, M.

Simoniello, P.

Thönnes, E.

Wiedemann, J.

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Inflammation-related response to irradiation in differenthuman skin culture systems∗J. Wiedemann1, P. Simoniello1, L. Madl1, E. Tho¨nnes1, M. Durante1,2, M. Podda3, and C. Fournier11GSI, Darmstadt, Germany; 2Technische Universita¨t Darmstadt, Germany; 3Dept. of Dermatology, Darmstadt HospitalIntroductionChronic inflammatory skin diseases (eczema, psoriasis)can be treated with low doses of irradiation [1]. Exposureto UV, photons and radon can alleviate the symptoms sug-gesting an anti-inflammatory effect. Apoptosis can be re-lated to this. The absence of an inflammatory response dur-ing apoptosis is not only due to lack of pro-inflammatorysignals, but apoptotic cells can actively suppress an inflam-mation by the release of anti-inflammatory cytokines likeTGF-β or IL-10 [2]. Changes induced by irradiation thatare potentially related to inflammation in skin will be in-vestigated.Materials and MethodsModel systems with different complexity-levels havebeen used: monolayer cultures of human primary ker-atinocytes (NHEK; Lonza), co-cultures of primary ker-atinocytes and fibroblasts (NHDF; Lonza) and human full-thickness skin equivalents (EFT400; MatTek; Ashland).Samples were irradiated comparing low versus high X-ray and C-ion (186 keV/μm) doses or UV-B intensities,fixed and processed 12 hours, 24 hours and 3 days afterirradiation for microscopic analysis and protein extraction.Hematoxylin & Eosin (H&E) staining and immunostainingagainst Caspase 3 and PARP have been performed. Cy-tokine release was quantified by ELISA.Results and DiscussionNo apoptosis was observed after X-ray and C-ion irra-diation in all model systems [3]. Cleaved caspase 3 andcleaved PARP could only be detected in monolayer cul-tures of NHEK after UV-B intensities of 40 and 60 mJ/cm2(Fig. 1A); the same intensities could not induce apoptosisin the more complex systems (data not shown). These sug-gest that apoptosis is not a trigger for the release of anti-inflammatory cytokines. C-ion irradiation induced mor-phological changes after exposure to a high dose; but ear-lier compared to UV-B (not shown). 3 days after UV-Birradiation (100 mJ/cm2), cobblestoned morphology of thebasal layer and a reduced cohesion of the stratum corneumoccur (Fig. 1B), indicating a radiation induced impair-ment of the differentiation process. The inflammatory re-sponse was assessed by detection of TGF-β and Il-1α re-lease in monolayer- and co-cultures. In unirradiated co-cultures the release of TGF-β and Il-1α is suppressed by∗Work supported by GREWIS No. 02NUK017A, DFG (GRK 1657)and HGS-HIRe.intercellular communication between NHEK and NHDF(data not shown) which is in agreement with the literature[4]. The enhancement of the pro-inflammatory cytokineIL1α in NHEK monocultures (data not shown) and co-cultures (fig.2) after exposure to moderate doses/intensitiesshows that the cytokine-balance is changed due to irradi-ation. Taken together, an induction of apoptosis was onlyobserved for UV-B exposure, not for ionizing irradiation.Modifications of the keratinocyte differentiation and re-lease of inflammatory factors was only observed for mod-erate and high doses of UV-B and ionizing radiation, butnot for low doses. In future experiments the influence ofimmune cells on the radiation response will be considered.ϭϬϬŵ:ͬĐŵϸ͖ϯĚĂǇϭϬϬŵ:ͬĐŵϸ͖ϯĚĂǇϲϬŵ:ͬĐŵϸ͖ϯĚĂǇWZWĨůĐů'W,ĂƐƉϯĨůĐůϭϮŚŽƵƌƐ ĂĨƚĞƌŝƌƌĂĚŝĂƚŝŽŶhsͲ΀ŵ:ͬĐŵϸ΁ Figure 1: (A) Western Blot analysis of NHEK cells af-ter UV-B irradiation. PC: positive control (HaCaT cells5 days; 10 Gy X-ray ); kDa: kilodalton; fl: full-length; cl:cleaved (B) H&E staining of tissue sections. circle: cab-blestoned morphology in the stratum basale (sb); arrows:reduced cohesion of stratum corneum (sc)Figure 2: IL1α and TGF-β release of co-cultures after irra-diation with x-ray and UV-BReferences[1] Rodel et al. (2002); Strahlenther Onkol 178:1–9[2] Voll et al. (1997); Nature 390:350–351[3] Simoniello et al. (2012). GSI Scientific Report. 432.[4] Le Poole et al. (1999); Br J Dermatol 140:409–416BIOPHYSICS-12 GSI SCIENTIFIC REPORT 2013214 doi:10.15120/GR-2014-1-BIOPHYSICS-12

Inflammation-related response to irradiation in different human skin culture systems

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Inflammation-related response to irradiation in different human skin culture systems

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