PHENOTYPIC ANALYSIS OF LYMPHOCYTE SUBPOPULATIONS IN LYMPH-NODES DRAINING THE EAR FOLLOWING EXPOSURE TO CONTACT ALLERGENS AND IRRITANTS

The murine local lymph node assay (LLNA) measures in vivo proliferatio n in draining lymph nodes (DLN) following topical exposure to chemical s to assess contact sensitization potential. However, proliferation ha s also been observed with some irritants. To further characterize even ts in the DLN during the LLNA and distinguish allergens from irritants , phenotypic analysis of lymphocyte subsets was made following topical exposure. In preliminary studies, mice were treated on the ears for 3 consecutive days, and 48 hr following the final application, analysis of CD3, CD4, CD8, and B220 expression was evaluated by flow cytometry . The allergens oxazolone (OXAZ) and picryl chloride (TNCB) and the ir ritant benzalkonium chloride (BC) increased cell number compared to ve hicle. The increase in lymph node cellularity for these materials was due to an increase in the total number of T and B lymphocytes. Interes tingly, even though contact sensitization is a cell-mediated immune re sponse (Th1), mice exposed to the contact allergens showed a preferent ial increase in B lymphocytes in the DLN as seen by an increase in the percentage of B220(+) cells. The percentage of B220(+) cells was 13.1 and 36.1% for OXA and TNCB, respectively, compared to percentages of 7.4 and 9.3% for irritant and vehicle, respectively. With some allerge ns, a concomitant decrease in the percentage of CD3(+) cells was seen. Time course studies demonstrated the increase in the percentage of B2 20(+) cells was seen in allergen treated mice by 24 hr after the final application of material, plateaued by 48 hr, and was still elevated b y 96 hr. In allergen-treated mice, percentages of B220(+) cells increa sed dose dependently. Further studies were performed to evaluate addit ional contact allergens and irritants and determine if evaluation of f low cytometric parameters could potentially identify contact allergens and differentiate them from irritants. Analysis of data from these st udies, which examined a total of five contact allergens and six irrita nts, showed that the modifications to the LLNA improved the identifica tion of irritants and allergens in individual experiments by including both phenotypic analysis of the DLN and cell number per node as endpo ints rather than either endpoint alone. (C) 1996 Society of Toxicology .