Examination of Phenotypic Changes in Peripheral Blood‐Derived Dendritic Cells Following Exposure to a Contact Allergen: Cell Surface Marker and Gene Expression

Abstract

The evaluation of the potential to induce allergic contact dermatitis is an important component of the overall safety assessment process of a new chemical entity that may be encountered through the skin. Currently, the murine local lymph node assay (LLNA) is the method of choice for assessing skin sensitization potential. However, despite the success of the LLNA as an alternative test method that results in a reduction in and refinement of animal usage, it does require the continued use of animals. Therefore, the development of an in vitro predictive test method for skin sensitization is still necessary. A number of different approaches have been taken to develop such a method including the examination of changes in cell surface marker expression in cultured human dendritic cells (DC). The purpose of this current study was to examine the effect of allergen exposure on peripheral blood mononuclear cell (PBMC)‐derived DC using a panel of cell surface markers known to be important in the development of allergic contact dermatitis; CD86, CD80, CD83, CD54, CD40, and CD1a. Changes in the expression of these markers were measured at the level of transcription and cell surface expression. The DC derived from the adherent cell fraction of human PBMC were exposed to either 1 mM or 5 mM dinitrobenzene sulfonic acid (DNBS), the water‐soluble analog of the strong contact allergen 2,4‐dinitrochlorobenzene for 24 hr. After treatment, surface markers expression was measured by flow cytometry, and total RNA was obtained for microarray analysis using Affymetrix U95Av2 Genechips®. Increases in both the cell surface expression and transcript levels of CD86 were observed for PBMC‐DC exposed to DNBS, with 5 mM DNBS inducing the greater change. Cell surface expression of CD80 and CD54 was similar for 1 mM DNBS treated and control cells, and no significant changes were seen at the transcript level. Treatment with 5 mM DNBS resulted in a decrease in cell surface expression for both of these markers. Although a significant allergen‐induced decrease in the transcript level for CD54 was noted, CD80 transcripts were increased. No marked effects on CD83 cell surface or gene expression were observed for either the 1 mM or 5 mM DNBS‐treated cells. Cell surface expression of CD40 and CD1a was somewhat variable in DC preparations treated with 1 mM DNBS, and no significant changes in transcript levels were induced. However, treatment with 5 mM DNBS induced a more marked decrease in cell surface expression of CD40 and CD1a and a significant decrease in the transcript level for CD1a. In summary, we found that changes in cell surface expression in PBMC‐DC following 24‐hr exposure to a contact allergen were reflective of the transcript levels for each of the markers examined with the exception of CD80 in cells treated with 5 mM DNBS.