Use of An in Vitro Skin Model for Determining Epidermal and Dermal Contributions to Irritant Responses

Abstract

We have used an in vitro skin model to study the responses of epidermal and dermal cells to irritants. This model consists of neonatal foreskin fibroblasts grown into a three-dimensional dermal structure on a nylon mesh. The dermal structure is seeded with keratinocytes and grown in calcium-containing medium at the air-liquid interface until a fully differentiated epidermis was formed. Phorbol-myristate acetate (PMA), a skin irritant, was incubated with this model. The PMA-conditioned culture medium was assayed for interleukin 1 alpha (IL-1 alpha) and tissue-type plasminogen activator (t-PA) by enzyme-linked immunosorbent assay (ELISA). Gelatin-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels were used to measure production of gelatinases. Exposure of the skin model to PMA resulted in the release of IL-1 alpha, t-PA, and gelatinases. This increase can be detected prior to a decrease in cell viability as measured by reduction of the mitochondrial dye MTT. Separation of the epidermis from the dermis and subsequent exposure of the dermis to PMA showed that in the presence of PMA the epidermis produces IL-1 alpha and the dermis alone produces t-PA. Both the epidermis and dermis produce gelatinases; only the epidermal gelatinase activities (72, 92 kd) are increased by PMA treatment. Since IL-1 alpha is an important regulator of immune and inflammatory responses and t-PA and gelatinase activities are elevated in many skin disorders, we believe that these assays in this in vitro skin model are useful for determining the contributions of epidermis and dermis to irritant responses.