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

Abstract

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 T_H1 cytokines (IL-2 and IFNγ) and T_H2 cytokines (IL-4 and IL-5) in lymph node cell cultures were significantly (P < 0.01) increased when DNCB was applied at the concentrations of 0.05 and 0.1%, respectively. As the DI was > 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.

Keywords::

• Local lymph node assay
• non-radioactive endpoints
• DNCB
• cytokine production