Evaluation of auricular lymph node cell lymphocyte proliferation and cytokine production as non-radioactive endpoints during murine contact allergy

The murine local lymph node assay (LLNA) has been developed as a test method to assess allergic contact dermatitis. In spite of the validity of the LLNA, attention was drawn to the two disadvantages: use of radioactivity for in vivo measurement of lymph node cell proliferation ([3H]-thymidine labeling) and the possibility of false positive results caused by non-specific cell activation as a result of inflammatory processes in the skin (irritation). We aimed to investigate the following non-radioactive endpoints of LLNA: 5-bromo-2′-deoxyuridine (BrdU) incorporation ex vivo and in vivo, in vivo and ex vivo cytokine production with or without phytohemagglutinin (PHA) stimulation. Here, 8-12-week-old female BALB/c mice were treated topically with the strong sensitizer 2,4-dinitrochlorobenzene (DNCB) in acetone:olive oil (AOO, 4:1 [v/v]) at levels of 0.025, 0.05, 0.01, or 0.25% (w/v). Ear thickness was also measured to determine the differentiation index (DI) indicating the proportion of non-specific activation due to irritating properties of test compound. At the concentration of 0.05%, stimulation index (SI) value was found to be 3 for DNCB based on in vivo and ex vivo BrdU incorporation. The results of the in vivo and ex vivo non-radioactive LLNA assays were compatible both with each other and with previous radioactive LLNA data. Our results indicate that non-radioactive endpoints may be used as an alternative to the [3H]-thymidine LLNA. The levels of TH1 cytokines (IL-2 and IFNγ) and TH2 cytokines (IL-4 and IL-5) in lymph node cell cultures were significantly (P 1, the applied concentrations of DNCB caused only allergic effect but not any irritant effect. This study reports that the use of these non-radioactive endpoints can assess allergic contact dermatitis caused by chemicals.