Cytokine endpoints for the local lymph node assay: Consideration of interferon-gamma and interleukin 12

The murine local lymph node assay (LLNA) is a method for the prospective id entification of contact allergens. Skin sensitization potential is assessed as a function of induced proliferative responses in lymph nodes draining t he site of topical exposure measured in situ by incorporation of radiolabel led thymidine ([H-3]thymidine). The results of previous investigations have demonstrated that the analysis of [H-3]thymidine incorporation represents a robust and reliable endpoint for the LLNA for the assessment of skin sens itizing activity for strong and moderate allergens and, in addition, many w eaker sensitizers. The aim of the current experiments was to explore the ut ility of the production of the cytokines interferon-gamma (IFN-gamma) and i nterleukin 12 (IL-12) by draining lymph node cells (LNC) as alternative rea douts for the LLNA. Animals were exposed to a range of skin sensitizers at two application conc entrations. The first of these was chosen on the basis of results from prev ious investigations to stimulate a strong proliferative response (tenfold o r greater increase in proliferation compared with concurrent vehicle contro ls). The second concentration of test material in each case was; the amount of chemical estimated to be necessary mathematically for elicitation of a stimulation index of 3 (EC3 value); the induction of a threefold or greater increase in proliferation is the current criterion for a positive response in the LLNA. In addition, analyses were conducted with para-aminobenzoic a cid (PABA), a non-sensitizing chemical shown previously not to induce LLNA responses. Secretion of IFN-gamma and the p40 subunit of IL-12 by draining LNC was measured by cytokine-specific enzyme-linked immunosorbent assay. In parallel experiments, LNC activity was assessed as a function of [H-3]thym idine incorporation ill situ. All the chemical allergens tested provoked robust proliferative responses, with the stimulation indices recorded at both test concentrations reflectin g only small changes in activity compared with previously recorded data. Ex posure to vehicle (4:1 acetone:olive oil, AGO) alone resulted in detectable , although variable, expression of both IFN-gamma and IL-12. Treatment with chemical allergen in each case caused a marked increase in IFN-gamma secre tion, with particularly vigorous production of cytokine being stimulated fo llowing exposure to oxazolone or hexyl cinnamic: aldehyde. In contrast, app lication of chemical allergens was not generally associated with elevated I L-12 p40 secretion. Exposure of mice to PABA did not result in increased IF N-gamma or IL-12 production compared with vehicle-treated controls. In gene ral, however, cytokine secretion did not correlate closely with the inducti on of LNC proliferation. These data indicate that expression by allergen-ac tivated LNC of IFN-gamma or IL-12 does not provide a reliable or sufficient ly sensitive endpoint for the LLNA compared with [H-3]thymidine incorporati on in situ. Copyright (C) 1999 John Wiley & Sons, Ltd.