In vivo UVB irradiation induces clustering of Fas (CD95) on human epidermal cells

In vitro studies with human cell lines have demonstrated that the death receptor Fas plays a role in ultraviolet (UV)-induced apoptosis. The purpose of the present study was to investigate the relation between Fas expression and apoptosis as well as clustering of Fas in human epidermis after a single dose of UVB irradiation. Normal healthy individuals were irradiated with three minimal erythema doses (MED) of UVB on forearm or buttock skin. Suction blisters from unirradiated and irradiated skin were raised, and Fas, FasL, and apoptosis of epidermal cells were quantified by flow cytometry. Clustering of Fas was demonstrated by confocal laser scanning microscopy on cryostat sections from skin biopsies. Soluble FasL in suction blister fluid was quantified by ELISA. Flow cytometric analysis demonstrated increased expression intensity of Fas after irradiation, with 1.6-, 2.2- and 2.7-fold increased median expression at 24, 48 and 72 h after irradiation, respectively (n = 4). Apoptosis was demonstrated by the TUNEL reaction, and the maximum of apoptotic cells was detected at 48 h after irradiation. Double-staining for Fas and TUNEL showed that apoptosis was restricted to the Fas-positive epidermal subpopulation, but there was no correlation between the intensities of Fas expression and TUNEL reaction. Median expression intensity of FasL-positive cells transiently decreased to 0.9- and 0.8-fold of the preirradiation respective level after 24 h and 48 h, respectively, and returned to the respective preirradiation level at 72 h after irradiation (n = 4). Concentrations of soluble FasL in suction blister fluid from UVB-irradiated skin did not differ from those in unirradiated skin (n = 5). Confocal laser scanning microscopy showed a rapid clustering of Fas within 30 min after irradiation. A simultaneous clustering of the adapter signalling protein FADD suggested that Fas clustering has a functional significance. Our results are in accordance with previous findings from in vitro studies, and suggest that Fas is activated in vivo in human epidermis after UVB exposure.