Research Grants 1997-1998

R3-Organotypic (Raft) Epithelial Culture System for Contact Dermatitis Testing

Craig Meyers, PhD
Pennsylvania State University, Hershey, Pennsylvania

The general dogma that skin is merely a protective covering of the body is rapidly changing. New information from in vivo and in vitro studies provides compelling evidence that the keratinocyte can initiate and actively participate in diverse inflammatory reactions. In response to environmental insult, keratinocytes independently release several types of immunoregulatory molecules. Additionally, keratinocytes express adhesion molecules immunoinflammatory associated with reactions, such as the intercellular adhesion molecule-1 (ICAM-1). keratinocytes can play a pivotal role in allergic Therefore, contact dermatitis (ACD) as well as irritant contact (ICD). We have developed a working hypothesis that an in dermatitis vitro keratinocyte culture system is assessing commercial and therapeutic products to induce ACD and ICD.

A crucial component of testing our working hypothesis is using a proper in vitro keratinocyte system. An assortment of techniques have been tried by various investigators to culture epithelial cells in culture. The organotypic (raft) culture system has been shown to most accurately mimic the in vivo physiology of the epidermis. Growing epithelial cells in the raft system has allowed for a "complete" differentiation program in contrast to what is achieved in monolayer cultures. We propose to evaluate the human epithelial raft culture system for its efficacy as a mechanistic system to test toxicity of commercial and therapeutic products. To accomplish this we will define the expression of immunoregulatory molecules in human epithelial raft culture tissues following treatment with a contact dermatitis inducing chemical. We propose that a set of indices can be defined based on the expression patterns of interleukin-1a (IL-1a), IL-aB, IL-6, tumor necrosis factor-a (TNF-a), granulocyte macrophage-colony stimulating factor (GM-CSF), transforming growth factor B (TGFB) and intercellular adhesion molecule-1 (ICAM-1) that will allow us to identify contact dermatitis-inducing compounds.

In Specific Aim 1 we will continue to define the expression of immunoregulatory molecules by human epithelial raft culture tissue. This will include:

Using keratinocyte lines derived from different donors.
Testing SLS and NS at continuous 10-fold dilutions to determine the sensitivity of the system.
Comparisons of organotypic epithelial tissues with keratinocyte monolayer cultures of cytokine expression levels both transcriptionally and translationally.
Beginning investigations with a second ICD inducing compound, phenol.
Begining investigations with a second SCD inducing compound, urushiol.
Continuing using immunohistochemistry to detect the spatial expression of the cytokines in the context of the three-dimensional tissue architecture.
Using ELISA assays to gain a better measurement of the expression levels of the cytokines.

Our second Specific Aim will be to define the expression of IL-1a, IL-1B, IL-6, TNF-a, GM-CSF, TGFB, and ICAM-1 following multiple exposures with dilutions of SLS and NS that exhibit little or no effect on the epithelial tissue when applied only once. Techniques used to analyze multiple exposed tissues will be the same as used for Specific Aim 1.

The third Specific Aim will be to determine if organotypic epithelial tissues can recover from exposure to dermatitis inducing agents and regain normal epithelial stratified and differentiated phenotype. First, tissues will be treated with SLS or NS as described but following treatment some of the test samples will be incubated for greater time periods allowing more time for recovery. Second, SLS or NS will be applied to only half the organotypic tissue. This is to determine if the untreated epithelium can enhance the ability of the treated half to recover a normal differentiation phenotype. Techniques used to analyze multiply exposed tissues will be the same as used for Specific Aim 1.

Finally in Specific Aim 4 we will continue to study changes in the human epithelial differentiation program as a result of ICD and ACD induction. In correlation with the continued investigations of cytokine expression as proposed in Specific Aim 1, we will also continue to correlate morphological and biochemical changes in the differentiation program. This will include cataloguing disturbances in the differentiation phenotype. As the dilutions of the toxicants become greater we will determine which disturbances in the differentiation phenotype are the most sensitive to insult. We will determine if phenol and urushiol also disturb the differentiation phenotype similar to SLS and NS or are there significant differences.