A modified murine local lymph node assay for the differentiation of contact photoallergy from phototoxicity by analysis of cytokine expression in skin-draining lymph node cells

Abstract

Since predictive differentiation of photoallergenic from phototoxic reactions, induced by low molecular weight compounds, represents a current problem, we tried to improve the differentiation between the two reactions by using a modified protocol of the local lymph node assay (LLNA). Briefly, groups of female BALB/c mice received compound solution or vehicle alone on the dorsum of both ears on 3 consecutive days. Immediately after compound application indicated groups of mice were exposed to a UVA light-dose of 10 J/cm2. Auricular lymph nodes draining the ear tissue were excised 24 h following the last exposure. Evaluation consisted of assessing lymph node weights and cell counts to monitor organ hyperplasia and in vivo-proliferative events following substance application. Furthermore, we analysed cytokine gene transcription in freshly prepared lymph node cells (LNC) and the cytokine release in vitro by restimulated CD4+ T-cells and antigen presenting cells (APC), both purified from the skin-draining lymph nodes. Both contact (photo) allergenic (oxazolone and tetrachlorosalicylanilide) and phototoxic substances (8-methoxypsoralen and acridine) caused a dose dependent increase in lymph node weights and cell counts pointing to an inflammatory process in the lymph nodes. Analysis of cytokine gene transcription ex vivo and cytokine release in vitro revealed that during the induction phase of contact (photo) allergy CD4+ T-cells produced IL-2 and IFN-γ as well as IL-4 and IL-10, whereas IL-6 was derived from APC. In contrast, phototoxic reactions caused only an upregulation of IL-2 and IFN-γ. Furthermore, we demonstrate that the release of IL-4 and IL-10 by CD4+ T-cells was clearly increased, whereas IL-6 and IFN-γ expression was reduced or not changed following a challenge with contact (photo) allergens revealing an allergy-indicative shift in cytokine expression. In conclusion, our results show that contact photoallergenic reactions could be differentiated from phototoxic events by analysis of LNC cytokine expression patterns.