The effects of UVA, mixed UVA + B, and solar-simulated irradiation were examined in human keratinocytes and melanocytes cultured in vitro. Irradiation with UVA, UVA + B, or the solar simulator caused a dose-dependent decrease in keratinocyte cell numbers and thymidine incorporation at 24 hours, with recovery after 48 and 72 hours. Divided dose regimens reduced the inhibitory effect of ultraviolet (UV) irradiation on cell numbers measured 24 hours after the last irradiation. Exposure to both UVA and UVA + B increased formation of cornified envelopes. Similar irradiance doses of UVA 80 minutes (1.12 J/cm2) and UVA + B 40 minutes (1.04 J/cm2) caused 2.4- and 3.3-fold increases in cornified envelope formation, respectively. With solar-simulated irradiation, the cornified envelope formation was increased by 3.5-fold after exposure of 8 minutes (2.6 J/cm2). Irradiation of melanocytes with UVA, UVA + B, or solar-simulated irradiation resulted in a dose-dependent decrease in melanocyte numbers after 24 hours compared with sham-irradiated controls. As a result of UV irradiation, tyrosinase activity of melanocytes measured at 24 hours was stimulated. UVA + B irradiation (1.04 J/cm2) increased tyrosinase activity approximately twofold, while UVA alone (1.1 J/cm2) increased tyrosinase four to sixfold and solar-simulated irradiation (1.3 J/cm2) increased tyrosinase approximately twofold compared to the control cells. Melanin content increased in cells after both UVA and mixed UVA + B irradiation. These results indicate that both UVA and mixed UVA + B irradiation had qualitatively similar effects on the proliferative and functional activity of skin-derived cells but that the type of irradiation and the dosage regimen affect the dose-response relationship.
The adaptive responses in skin to ultraviolet (UV) radiation include increased cornification of keratinocytes and increased synthesis and distribution of melanin by melanocytes.The possible involvement of paracrine factors in the generation of these responses was studied in a novel twostage culture model. Human melanocytes or keratinocytes were first irradiated or sham-irradiated and then the conditioned media collected from these cells after 24 h were used to treat unirradiated skin cells.Immunofluorescent staining for transforming growth factor (TGF)-1 was increased in UV-irradiated keratinocytes compared with sham-irradiated cells. Increased TGF-1 was also detected in the culture media of irradiated keratinocytes. Treatment of unirradiated keratinocytes with conditioned media collected from UVirradiated keratinocytes resulted in increased absolute numbers and percentages of cornified envelopes per well compared with treatment with conditioned media from sham-irradiated keratinocytes. The magnitude of this effect increased with increased dose of initial irradiation. The effects of conditioned media from UVR-treated cells were mimicked by authentic TGF-1. Treatment of conditioned media from irradiated cells with an antibody shown to neutralise the effects of TGF-1 but not with a non-immune antibody of similar isotype, abolished this bioactivity of the conditioned media from UV-irradiated cells. Immunofluorescent staining for ACTH was also increased in UV-irradiated keratinocytes. Conditioned media from UV-irradiated keratinocytes increased tyrosinase activity of unirradiated melanocytes, an effect which was mimicked by authentic ACTH. This bioactivity of conditioned media from irradiated keratinocytes was abolished in the presence of an antibody which neutralised the activity of ACTH but not MSH. These results provide evidence to support the involvement of TGF-1 and ACTH in the cornification and pigmentary responses respectively of skin cells after UV exposure.
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