Introduction: The murine local lymph node assay (LLNA) was developed as an alternative to guinea pig models for the assessment of the xenobiotic contact sensitization potential. However, it would be advantageous to have an alternative endpoint to the usual radioisotopic-dependent measures. In the present study, we investigated the development of a nonradioisotopic endpoint for LLNA using immunohistochemistry.
Methods: Female Balb/c mice were treated by the topical application of strong sensitizers, 2,4-dinitrochlorobenzene (DNCB) and toluene diisocyanate (TDI), and a strong irritant, sodium lauryl sulfate (SLS), on the dorsum of both ears once daily for three consecutive days. The proliferation of cells in the auricular lymph node and ears was analyzed by means of the labeling index (LI) of bromodeoxyuridine (BrdU) incorporation into cells.
Results: Skin reactions, consisting of increased ear thickness and the presence of inflammatory cell infiltrates, were observed in mice treated with DNCB and TDI. The cell number and the weight of the lymph nodes in the mice treated with the allergens, DNCB and TDI, were increased compared to vehicle control. We observed an increase in the areas of the B220(+) cells in the lymph nodes of mice treated with allergens, as determined by immunohistochemistry. There was an increase in the percentage of B220(+) cells in mice treated with DNCB and TDI compared to the vehicle control, but not in those treated with SLS. Because we observed an increase in the percentage of B cells in the allergen-treated group, we measured the stimulation index (SI) in the cortex and medulla (C+M) of the lymph node. The SI values of the C+M in the lymph nodes of the mice treated with DNCB and TDI were increased more than threefold compared with that of the control. However, the SI of the C+M in the lymph nodes of the mice exposed to 25% SLS was not significantly increased compared to the vehicle control, although the lymph node weight of the SLS group was significantly increased.
Discussion: In Balb/c mice, BrdU immunohistochemistry showed its potential use for the identification and differentiation of chemicals with the capacity to induce irritation and sensitization. The results suggest that the measurement of the SI in the cortex and medulla of the lymph node using BrdU immunohistochemistry could provide a useful method to screen irritants and allergens.