A plasmacytoid dendritic cell (CD123+/CD11c-) based assay system to predict contact allergenicity of chemicals

Abstract

A predictive allergenicity test system for assessing the contact allergenicity of chemicals is needed by the cosmetic and pharmaceutical industry to monitor product safety in the marketplace. Development of such non-animal alternative assay systems for skin sensitization and hazard identification has been pursued by policy makers and regulatory agencies. We investigated whether phenotypic and functional changes to a subset of dendritic cells (DC), plasmacytoid DC (pDC), could be used to identify contact allergens. To achieve this goal, normal human DC were generated from CD34+ progenitor cells and cryopreserved. Frozen DC were thawed and the pDC fraction (CD123+/CD11c-) was harvested using FACS sorting. The pDC were cultured, expanded, and exposed to chemical allergens (N=26) or non-allergens (N=22). Concentrations of each chemical that resulted in >50% viability was determined using FACS analysis of propidium iodide stained cells using pDC from 2 to 5 donors. Expression of the surface marker, CD86, which has been implicated in dendritic cell maturation, was used as a marker of allergenicity. CD86 expression increased (> or =1.5-fold) for 25 of 26 allergens (sensitivity=96%) but did not increase for 19 of 22 non-allergens (specificity=86%). In a direct comparison to historical data for the regulatory approved, mouse local lymph node assay (LLNA) for 23 allergens and 22 non-allergens, the pDC method had sensitivity and specificity of 96% and 86%, respectively, while the sensitivity and specificity of the LLNA assay was 83% and 82%, respectively. In conclusion, CD86 expression in pDC appears to be a sensitive and specific indicator to identify contact allergenicity. Such an assay method utilizing normal human cells will be useful for high throughput screening of chemicals for allergenicity.

Figure 1

Representative transmission electron microscopy micrograph showing expression of Birbeck granules in pDC

Figure 2

FACS sorting and characterization of dendritic cells. a) FACS sorting of DC/LC into pDC (CD123+ / CD11c-) and mDC (CD123- / CD11c+); b) Morphology of pDC and mDC following a five day culture period. mDC are more dendritic than pDC as observed using light microscopy (40× objective), and c) Comparison of surface marker expression of pDC and mDC.

Figure 3

Expansion of pDC obtained from FACS sorted dendritic cells. pDC were cultured in pDC-MM medium and were counted every 2-3 days. Results of two independent experiments indicate that pDC can be expanded by 12-32 fold.

Figure 4

FACS analysis showing CD86 expression by pDC following 24 hour exposure to allergens, hydroquinone (HQ), Balsam of Peru (BP), nickel sulfate (Ni), and cinnamaldehyde (CA) and non-allergens, propylene glycol (PG), Tween 20 (TW), and ethanol (ET). After exposure, pDC were washed, labeled with FITC-conjugated anti-CD86 monoclonal antibodies, and analyzed for CD86 expression using FACS. PBS treated cells were used as negative controls.