Local Lymph Node Assay: Differentiating Allergic and Irritant Responses Using Flow Cytometry

The murine local lymph node assay (LLNA) is a method for assessing the contact sensitization potential of chemicals. Based on events that occur during the induction phase of a contact sensitization response, the LLNA measures the in vivo proliferation of cells in the draining lymph nodes (DLNs) of mice following topical exposure to chemicals. In terms of predictive identification of important skin sensitizers, the LLNA has been shown to be at least as sensitive as, and much more reliable than, current guinea pig tests. However, proliferation has also been observed following treatment with some irritants. In an attempt to distinguish allergic from irritant-induced proliferation, flow cytometric techniques have been used to examine the phenotype of lymphocyte subsets in the DLNs as well as markers of T-lymphocyte activation and memory. Mice were treated on the ears for 3 consecutive days with allergens or irritants. The DLNs were harvested 72 h after the final treatment. Single-cell suspensions were prepared, counted, and stained for analysis of the percentages of T cells and B cells and T-cell expression of two adhesion molecules that have been associated with differentiating naïve and activated/memory T cells, CD62L (L-selectin) and CD44 (H-cam). Increases in lymph node cellularity were observed in both allergen- and irritanttreated mice relative to naïve and vehicle-treated animals. Mice treated with allergens showed a preferential increase in the percentage of B220+ B cells compared with irritant-treated mice. Treatment with allergens, but not irritants, resulted in a selective increase in the percentages of CD4+ and CD8+ cells expressing the T-cell activation/memory phenotype CD62LloCD44hi. Taken together, flow cytometric analysis of cell phenotype and expression of T-cell activation/memory markers may provide important information for differentiating allergen- and irritant-induced proliferative responses in the DLNs of chemically treated mice.