IN VITRO MODEL FOR IMMUNOTOXICITY: SURFACE MARKER EXPRESSION AND CYTOKINE RELEASE IN NORMAL HUMAN DENDRITIC CELLS (DC-100).

Dendritic cells (DC) play a key role in the initiation of immune response by recognizing, internalizing, processing, and presenting antigens to T cells. However, DC function can be altered by environmental factors, pharmaceutical agents, and exogenous chemicals. These perturbations to immune cell function, i.e. immunotoxicity, can have significant effects on the natural defense mechanisms that ward off pathogens.   While animal based assays are commonly used for immunotoxicity assessment, the Cosmetic Directive and REACH legislation bans the use of animals for many such studies.  To examine the potential use of DC for immunotoxicity evaluation, large numbers of DC were generated from CD34+ progenitor cells and characterized for their expression of HLA-DR, CD1a, langerin, Birbeck granules, and co-stimulatory molecules (e.g. CD80, CD83, CD86, and CD40).  In this study, we evaluated the effect of 5 immunotoxic compounds (ITC) on DC function.  DC functionality was monitored by measuring surface marker expression and cytokine secretion following exposure to lipopolysaccharide (LPS).  DC were first exposed to non-cytotoxic concentrations of the ITC for 24 hr and then to LPS for an additional 18 hr.  FACS analysis of ITC-exposed DC showed effects in the rank order of immunotoxicity (severe to low effect): Tributyltin chloride > Cyclosporine A > Benzo(a)pyrene > Fursemide and Urethane. Dose-dependent decreases in the secretion of immuno-stimulatory cytokines including interleukin (IL)-12, IL-6, and IL-1β were observed. Conclusion: The release of immunostimulatory cytokines may serve as an endpoint to predict immunotoxicity. Due to concerns with animal models in terms of cost, ethical issues, and relevance to hazard assessment in humans, the DC based assay could be an attractive in vitro model to predict immunotoxicity of compounds.

Birbeck granules, CCR-7, CD83, CD86, IL-12, IL-1β, IL-6, Immuno-stimulatory cytokines, Immunotoxicity, MIP-3α, Rantes, TNF-α

Benzo(a)pyrene, Cyclosporine, Fursemide, Lipopolysaccharide (LPS), Tributyltin chloride, Urethane, Verapamil