title: 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 ([(3)H]-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 [(3)H]-thymidine LLNA. The levels of T(H)1 cytokines (IL-2 and IFNγ) and T(H)2 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.
authors.affiliation: Faculty of Pharmacy, Department of Toxicology, Ankara University, Ankara, Turkey.
authors.forename: Ozge Cemiloglu
authors.initials: OC
authors.lastname: Ulker
authors.forename: Aysegul
authors.initials: A
authors.lastname: Atak
authors.forename: Ilker
authors.initials: I
authors.lastname: Ates
authors.forename: Asuman
authors.initials: A
authors.lastname: Karakaya
mesh_terms: D000818:Animals; D049109:Cell Proliferation; D002478:Cells, Cultured; D016207:Cytokines / Q000276:immunology*; D003876:Dermatitis, Atopic / Q000139:chemically induced / Q000276:immunology* / Q000473:pathology; D004137:Dinitrochlorobenzene / Q000009:adverse effects / Q000494:pharmacology; D005260:Female; D007509:Irritants / Q000009:adverse effects / Q000494:pharmacology; D008198:Lymph Nodes / Q000276:immunology* / Q000473:pathology; D051379:Mice; D008807:Mice, Inbred BALB C; D012867:Skin / Q000276:immunology / Q000473:pathology; D018417:Th1 Cells / Q000276:immunology* / Q000473:pathology; D018418:Th2 Cells / Q000276:immunology* / Q000473:pathology
chemical_list: D016207:Cytokines; D004137:Dinitrochlorobenzene; D007509:Irritants