Profiling the Response of Human Hair Follicles to Ultraviolet Radiation  Zhongfa Lu, Tobias W. Fischer, Sybille Hasse, Koji Sugawara, York Kamenisch, Sven Krengel, Wolfgang Funk, Mark Berneburg, Ralf Paus  Journal of Investigative Dermatology  Volume 129, Issue 7, Pages 1790-1804 (July 2009) DOI: 10.1038/jid.2008.418 Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Increased catagen induction and LDH release, and reduced hair shaft elongation with increasing doses of UVB irradiation. Hair follicles were irradiated with either 20 or 50mJcm-2 over a period of 9 days. Catagen development (a and b) and hair shaft elongation reduction (c and d) of 72 hair follicles was measured and the results are summarized graphically. After day 5 in culture, there was a linear and significant increase of catagen induction (*P<0.05) and hair shafts grew significantly less compared with control (*P<0.05; #P<0.001) when irradiated with 50mJcm-2 (d), whereas there was only a minor, nonsignificant reduction in hair shaft elongation after 20mJcm-2 UVB (c). There was a strong and significant increase of LDH in the supernatant of follicles irradiated with 50mJcm-2 as early as day 5 (*P<0.05) with increasing significance at later time points (#P<0.001) (e). Arrowheads mark the days of UVB exposure (days 1, 3, and 5). Journal of Investigative Dermatology 2009 129, 1790-1804DOI: (10.1038/jid.2008.418) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 UVB exposure–induced suppression of proliferation and increase of apoptosis in hair matrix keratinocytes. 72 hair follicles were analyzed for apoptosis (TUNEL) and proliferation (Ki67) after 9 days in culture and UVB irradiation (20 or 50mJcm-2). Of each group, one representative photograph of the immunofluorescence for DAPI, TUNEL, and Ki67 is displayed (a–i). Quantitative analyses revealed that Ki67 was positive in 22% of cells in a defined area of the bulb region in nonirradiated follicles (c and j) versus 1.36 and 0.76% in irradiated hair follicles (20 and 50mJcm-2 UVB, respectively; j). Cells were TUNEL+ in 2.5% per defined reference area in nonirradiated follicles (b and j) versus 27% (20mJcm-2; e and j) and 57% (50mJcm-2; h and j) in UVB-exposed follicles. DP, dermal papilla; MK, matrix keratinocytes; CC, cortical cells; IRS, inner root sheath; ORS, outer root sheath; CTS, connective tissue sheath. Scale bar=100μm in panels (a–i). Journal of Investigative Dermatology 2009 129, 1790-1804DOI: (10.1038/jid.2008.418) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Casp-3 is activated in matrix keratinocytes at 20mJcm-2, but not at 50mJcm-2, although detectable at 50mJcm-2 in the inner root sheath. Controls (nonirradiated hair follicles) show no immunoreactivity (IR) for casp-3 (a). Casp-3-IR is observed at day 9 in single matrix keratinocytes of hair follicles that had been irradiated with 20mJcm-2 (b), whereas at 50mJcm-2 (c), no casp-3-IR is expressed in matrix keratinocytes. However, at 50mJcm-2, casp-3-IR is observed in the inner root sheath (c). Representative pictures of two independent experiments are shown. Negative control is displayed in (d). DP, dermal papilla; MK, matrix keratinocytes; casp-3-immunoreactivity (green-fluorescence) is indicated by white arrows. Scale bar=30μm in whole panel. Journal of Investigative Dermatology 2009 129, 1790-1804DOI: (10.1038/jid.2008.418) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 p53 is strongly induced in matrix keratinocytes around the dermal papilla and along the Henle's layer of the inner root sheath at 20mJcm-2, but it is expressed less at 50mJcm-2. Immunoreactivity for p53 was strongly detected in matrix keratinocytes at 20mJcm-2 (b) compared to controls (a), whereas at 50mJcm-2 (c) expression in matrix keratinocytes was lower (versus 20mJcm-2), but still higher than in sham-irradiated controls. Representative pictures of three independent experiments are shown. Negative control is displayed in (d). DP, dermal papilla; MK, matrix keratinocytes; p53-immunoreactivity (green-fluorescence) is indicated by white arrows. Scale bar=100μm in whole panel. Journal of Investigative Dermatology 2009 129, 1790-1804DOI: (10.1038/jid.2008.418) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Mitochondrial common deletion was induced on 20mJcm-2. An increased rate of mutations of mitochondrial (mt) DNA as represented by the 4,977 base-pair large-scale deletion, the so-called common deletion, was detected in human hair follicles after UV irradiation with 20mJcm-2 by real-time PCR. Journal of Investigative Dermatology 2009 129, 1790-1804DOI: (10.1038/jid.2008.418) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Oxidatively damaged genomic DNA is detected at 20mJcm-2, but not at 50mJcm-2. Oxidative DNA damage is detected by immunohistochemistry using antibody against 8-OH-dG in matrix keratinocytes and large proximal parts of the inner root sheath on irradiation with 20mJcm-2 (d and e), whereas at 50mJcm-2 (g and h), the detection is very low and restricted to the middle parts of the matrix keratinoyte horns. Two selected hair follicles per condition (0, 20, 50mJcm-2) from three independent experiments are presented. Unirradiated control is displayed in a and b; negative controls for each condition are displayed in c, f and i. DP, dermal papilla; MK, matrix keratinocytes. Scale bar=100μm in whole panel. Journal of Investigative Dermatology 2009 129, 1790-1804DOI: (10.1038/jid.2008.418) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Melanin content strongly declined in UVB-irradiated hair follicles. Masson–Fontana stain revealed a strong decline of the melanin content in hair follicles exposed to both UVB doses compared with controls after 9 days in culture. Representative photographs show signs of hair follicle dystrophy, such as abnormally large and ectopically located melanin granules at both doses, 20mJcm-2 (b) and 50mJcm-2 (d), which are not present in nonirradiated control (a and c). UVB exposure significantly suppressed melanin positivity as assessed by quantitative densitometric evaluation (*P<0.01) at both UV-doses, but, surprisingly, there was no significant difference between the 20mJcm-2 group and the 50mJcm-2 group (e). DP, dermal papilla; MG, melanin granula. Scale bar=50μm in whole panel. Journal of Investigative Dermatology 2009 129, 1790-1804DOI: (10.1038/jid.2008.418) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Immunoreactivity of TGFβ2 and ACTH changes in response to UVB irradiation although α-MSH is not affected. In control hair follicles, TGFβ2-immunoreactivity (IR) was observed in the outermost area of outer root sheath cells (ORSC) (a), which shifted to the cuticle of the inner root sheath on irradiation by UVB (b and c). ACTH-IR was positively expressed in ORSC, connective tissue sheath (CTS) cells as well as in bulb cells of human hair follicles (d). After UVB irradiation at both doses, 20mJcm-2 (e) and 50mJcm-2 (f), ACTH expression was significantly less expressed than in controls (*P<0.01) (j). Interestingly, the expression of α-MSH was very weak and did not significantly differ among the control, 20, and 50mJcm-2 groups (g–i). M, medulla; C, cortex; CC, cortical cells; IRS, inner root sheath; CIR, cuticle of the inner rooth sheath; Hx, Huxley's layer; He, Henle's layer; ORS, outer root sheath; CTS, connective tissue sheath; DP, dermal papilla; MK, matrix keratinocytes. Scale bar=50μm in panels (a–d and g, h); 100μm in panels (e, f and i). Journal of Investigative Dermatology 2009 129, 1790-1804DOI: (10.1038/jid.2008.418) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 Quantitative analyses of nongranulated and degranulated mast cells in the CTS of human hair follicles after UVB irradiation. The total number of mast cells as well as the number of degranulated mast cells in the CTS increased after UVB irradiation as detected in Giemsa stained hair follicle sections (magnification × 400; a–f). Quantitative analyses revealed a dose-dependent increase in the absolute number of nondegranulated mast cells and degranulated mast cells (g). The relative percentage increase of degranulated mast cells is displayed in (h). Although 20mJcm-2 caused a significant difference in the control group, this was further promoted by irradiation with 50mJcm-2 UVB (*P<0.01, #P<0.05). Furthermore, compared to controls (b), the granules became bigger and acidophilic in a dose-dependent manner as assessed by Giemsa-stained sections of human hair follicles (d and f). Scale bar=50μm in panels (a, c and e); 10μm in panels (b, d and f). Journal of Investigative Dermatology 2009 129, 1790-1804DOI: (10.1038/jid.2008.418) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 10 Quantitative analyses of c-Kit+ cells on UVB irradiation. c-Kit (CD117) immunofluorescence showed most predominant staining in the CTS, but also a cobblestone-like pattern above the dermal papilla, representative of hair matrix keratinocytes on irradiation with 50mJcm-2 (d) compared to control (c). However, the number of c-Kit+ cells revealed no striking difference on UVB irradiation, whereas intensity of c-Kit immunoreactivity in the epithelial hair matrix was upregulated by UVB (d), which was quantified with NIH image software (e) (*P<0.05). Counterstaining for cell nuclei was performed with DAPI (a and b). DP, dermal papilla; MK, matrix keratinocytes; CTS, connective tissue sheath; C, cortex; M, medulla. Scale bar=50μm in whole panel. Journal of Investigative Dermatology 2009 129, 1790-1804DOI: (10.1038/jid.2008.418) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 11 Low UVB (20mJcm-2) induces apoptosis, whereas medium UVB dose (50mJcm-2) induces necrosis in hair follicle matrix keratinocytes. It is hypothesized that after UV-induced death-receptor-mediated induction of casp-8 and/or mitochondrial damage mediated induction of casp-9, the effector caspase casp-3 is activated after irradiation of human hair follicles with 20mJcm-2. At the same UV-dose, further apoptosis-related events such as p53 activation and DNA fragmentation (TUNEL) are observed as well as mitochondrial common deletion (mtCD) and oxidatively damaged genomic DNA (8-OH-dG) (a). At the higher dose of 50mJcm-2, casp-3, p53, mtCD and 8-OH-dG are not or are less observed, whereas TUNEL+ matrix keratinocytes and LDH levels in supernatants are significantly increased, indicative of increasing necrotic cell death (b). Journal of Investigative Dermatology 2009 129, 1790-1804DOI: (10.1038/jid.2008.418) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions