Cytokine mRNA expression in an in vitro human skin model, SKIN(2).

SKIN(2) ZK1300 is a three-dimensional human skin model consisting of multilayered dermal fibroblasts and well-differentiated epidermal keratinocyte layers, including a stratum corneum. To characterize this model better, constitutive levels of cytokine gene expression were determined. Reverse transcriptase-polymerase chain reaction (RT-PCR), followed by liquid hybridization to labelled internal probes, demonstrated that interleukin (IL)-1I, IL-1beta, IL-6, IL-8, IL-10, tumour necrosis factor (TNF)I, granulocyte macrophage-colony stimulating factor (GM-CSF), transforming growth factor (TGFbeta1) and IL-12 p35 mRNAs were constitutively expressed whereas IL-12 p40 was not. The contribution of the dermal component of this human skin model (Model ZK1100) was further characterized by determining constitutive cytokines expressed and their modulation by phorbol 12-myristate, 13-acetate (PMA). The dermal component, consisting of multilayered human dermal fibroblasts, constitutively expressed message for IL-1I, 1L-1beta, IL-6, IL-8, TGFbeta1, GM-CSF and IL-12 p35. Message was not detected for IL-10, TNFI or IL-12 p40. PMA treatment of the multilayered dermal fibroblasts increased steady-state mRNA levels of IL-1I, IL-1beta, IL-6, IL-8, GM-CSF and TGFbeta1, but did not induce IL-10, TNFI or IL-12 p40 expression at the dose and times tested. In summary, these studies demonstrate that the SKIN(2) three-dimensional human skin cultures, and their dermal component, constitutively express mRNA for an array of inflammatory and immunomodulatory cytokines, and that PMA exposure modulates mRNA levels of the dermal cytokines.

Quantitative in vitro assessment of phototoxicity using a human skin model, Skin2.

The ability to accurately predict the phototoxic potential of personal and skin care products remains a key element in assessing the safety of premarketed products. To find a reliable in vitro alternative test for photoirritancy, the European Commission and the European Cosmetic Association are conducting a 3-year, European validation study. Based on the results of this study, an in vitro photoirritancy method will be selected for incorporation into new international guidelines for photoirritancy testing. As a part of this study, Skin2, a cultured human skin system, was used to evaluate the phototoxic potential of chemicals with known photoirritative properties. The Skin2 ZK1351, a 3-dimensional co-culture system, consists of dermal fibroblasts and a multilayered epidermis comprising differentiated keratinocytes. This product line has previously been used to evaluate the irritative potential of topically applied ingredients and products. In this study, various concentrations of the test chemicals were applied to the epidermal side of the Skin2 tissue for contact times of 1 h or 24 h and then the tissue was exposed to 2.9 J/cm2 of ultraviolet A (UVA) radiation. Treated but nonirradiated tissues were also assayed to predict the cytotoxic potential of the test chemicals, which could mask the phototoxic reaction. After exposure, the tissue substrates were rinsed free of test chemicals and allowed to recover for 24 h. Following this incubation, the MTT reduction assay was used to assess cytotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Skin(2TM): An in vitro model to assess cutaneous immunotoxicity.

The skin serves as a complex barrier protecting the internal environment against external factors (e.g. bacteria, viruses, environmental toxins and UV light). For the epidermis to produce the most effective toxicological, immunological and biochemical barrier, the major cell types of the epidermis (i.e. keratinocytes) and in the dermis (i.e. fibroblasts) must function together in a dynamic integrated fashion. Furthermore, epidermal-dermal intercellular biochemical signals such as interleukins (IL), cytokines and other growth factors provide the skin with local homoeostatic signals to ensure the skin's immune integrity in response to a variety of environmental insults. Other investigators have shown that exposure of the skin to long-wave ultraviolet light (UVA) and mid-range ultraviolet light (UVB) can alter epidermal immune functions, including epidermal cytokine (e.g. IL-1, IL-6, TNF-alpha, IL-10 and GM-CSF) levels. The studies reported here use a co-culture system of dermal fibroblasts and well differentiated epidermal layers with an attached stratum corneum to form an in vitro human skin analogue. Baseline endogenous levels of IL-1alpha and tumour necrosis factor-alpha (TNF-alpha) were detected by using commercially available ELISA kits. The tissue substrates were exposed to UVA/UVB light (280-400 nm). The UV light was administered by a Dermsol 3 mercury halide solar simulator configured with filters to remove energy levels below 280 nm and above 410 nm. Skin tissue irradiated at 4J/cm(2) revealed a significant increase in IL-1alpha and TNF-alpha in comparison with non-UV irradiated tissue. Additional experiments revealed that the topical administration of indomethacin (0.1 to 10mg/ml) to the skin tissue ameliorated the up-regulation of these immune cytokines following UV irradiation. The use of such an in vitro co-culture system may provide researchers with a unique method to quantify mechanistically immunotoxicological events in the skin after exposure to ultraviolet light.