Keratinocytes act as a source of reactive oxygen species by transferring hydrogen peroxide to melanocytes

Basal hydrogen peroxide (H2O2) levels in normal human epidermal keratinocytes (NHEK) and melanocytes (mel) were compared on a per cell basis and found to be significantly higher in keratinocytes. Since H2O2 is a neutral molecule capable of permeating through cellular membranes, we then investigated the possibility that H2O2 transfer might occur between these two types of cells. Because the ratio of keratinocytes to mel in skin is 36:1, keratinocytes may act as a source of reactive oxygen species (ROS) even by passive diffusion and, thus, affect melanocytic functions. In order to measure H2O2 transfer, a fluorescence-based co-culture system was developed in which mel were first pre-labeled with 5-(and-6)-chloromethyl-2',7'-dichlorodihydro-fluorescein diacetate (DCFdA). When mel were co-cultured with keratinocytes, fluorescence increased as a function of keratinocyte cell number. Thus, for mel incubated with 1-, 1.5-, and 2-fold the number of keratinocytes, fluorescence increased by 22.6% (+/- 2.8%), 25.6% (+/- 4.8%), and 39.9% (+/- 4.1%), respectively. Separating the cells with a transwell membrane did not prevent the transfer, whereas the addition of catalase to media significantly reduced the transfer of H2O2 to mel. In conclusion, keratinocytes appear to be a previously unexamined source of ROS that may affect neighboring skin cells, such as mel, and, as a result, may influence the process of melanogenesis or contribute to the progression of vitiliginous lesions.