Elucidating changes in surface marker expression of dendritic cells following chemical allergen treatment

Dendritic cells (DC) are highly specialized antigen-presenting cells (APC) located in lymphoid and many nonlymphoid tissues, and Langerhans cells (LC), a specialized form of DC, are found in the skin. LC play a critical role in the induction of contact dermatitis and therefore have become a focal point for the development of in vitro cell-based methods for contact sensitization testing. Because of the low abundance of skin-derived LC, methods to culture DC from peripheral blood are being used by investigators to generate LC surrogates to examine the effects of sensitizing chemicals on APC. It has been reported recently that chemical allergens can induce changes in the expression of various DC surface markers and it has been suggested that the measure of these changes in surface marker expression following allergen treatment could provide the basis for an in vitro test method to predict the contact sensitization potential of a chemical. For the work presented here, DC were differentiated from human peripheral blood mononuclear cells (PBMC-DC) in culture medium containing GM-CSF and interleukin (IL)-4 to ensure an immature phenotype or were derived from the KG-1 cell line (KG-1 DC) using a defined cytokine cocktail consisting of GM-CSF, IL-4, Flt-3/Flk-2-ligand, thrombopoietin, stem cell factor, and tumor necrosis factor-alpha (TNFalpha). Surface marker expression (HLA-DR, CD54, CD80, and CD86) on these DC was measured by flow cytometry after 48 h treatment with the known chemical allergens dinitrofluorobenzene (DNFB) and methylchloroisothiazolinone/methylisothiazolinone (MCI/MI), the irritant sodium dodecyl sulfate, lipopolysaccharide (LPS), and TNFalpha. Treatment of PBMC-DC with either MCI/MI or DNFB induced a slight upregulation of class II major histocompatibility (MHC) expression (HLA-DR), whereas LPS and TNFalpha significantly upregulated CD54 and slightly upregulated CD80 and HLA-DR expression. For KG-1 DC, only MCI/MI upregulated CD86 expression, whereas TNFalpha upregulated CD54 and slightly upregulated CD80 and CD86 expression. SDS had no effect on surface marker expression in either PBMC-DC or KG-1 DC. Changes in surface marker expression in PBMC-DC treated with chemical allergens were detected in two of five donors, suggesting a limited sensitivity of PBMC-DC under these defined isolation and culture conditions. Furthermore, we found that the presence of GM-CSF and IL-4 during chemical allergen treatment masked the ability to detect changes in surface marker expression. Our data suggest that, under these culture and treatment conditions, measurement of surface marker changes in vitro using PBMC-DC or KG-1 DC does not provide a sensitive in vitro method with sufficient dynamic range for assessing the contact sensitization potential of a chemical.