Local lymph node assay: study of the in vitro proliferation and control of the specificity of the response by FACScan...

The murine local lymph node assay (LLNA) has been proposed as a screening procedure to identify contact allergens (Kimber, Hilton and Weisenberger, Contact Dermatitis 1989, 21, 215; Kimber and Weisenberger, Archives of Toxicology 1989, 63, 274). In some cases irritants have given rise to proliferative responses and it is of interest to investigate whether these responses differ in the type of cells involved. We have studied the proliferative response in vitro to topically applied sodium lauryl sulphate (SLS) and 2,4-dinitrochlorobenzene (DNCB). The test chemical or vehicle alone was applied for 3 consecutive days to the dorsum of both ears of Balb/c strain mice at three different concentrations, the highest concentration being the maximum non-irritating concentration (MNIC). Cell cultures were made 72 hr after the final exposure: draining auricular lymph nodes were excised and a suspension of lymph node cells (LNC) was prepared. Cellularity (total number of LNC/animal) and proliferative activity were assessed; proliferation was measured by culture of LNC for 24 hr with [(3)H]thymidine. LNC were also studied by FACScan analysis: cells were incubated with fluorescein isothiocyanate (FITC)-labelled monoclonal antibodies against the T-cell marker CD3 and the activated T-cell marker CD25 (IL2 receptor). In the case of DNCB dose-response curves were obtained for both cellularity and proliferative response in comparison with the controls: there was a strong increase in both parameters for the MNIC.SLS, a non-sensitizing skin irritant, induced a much lower response, slightly increased at the MNIC in comparison with the controls. By FACScan analysis we measured the rates of CD3- and CD25-positive cells in the LNC. No significant difference was obtained for SLS in comparison with the controls. In the case of DNCB, there was a significant increase in CD3-positive cells and a large increase in CD25-positive cells in comparison with the controls and SLS. These parameters could be of great interest to help distinguish between contact sensitizers and irritants. We are presently investigating other irritants and sensitizers.