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 identification of contact allergens. Skin sensitization potential is assessed as a function of induced proliferative responses in lymph nodes draining the site of topical exposure measured in situ by incorporation of radiolabelled thymidine ([3H]thymidine). The results of previous investigations have demonstrated that the analysis of [3H]thymidine incorporation represents a robust and reliable endpoint for the LLNA for the assessment of skin sensitizing activity for strong and moderate allergens and, in addition, many weaker sensitizers. The aim of the current experiments was to explore the utility of the production of the cytokines interferon-gamma (IFN-gamma) and interleukin 12 (IL-12) by draining lymph node cells (LNC) as alternative readouts for the LLNA. Animals were exposed to a range of skin sensitizers at two application concentrations. The first of these was chosen on the basis of results from previous investigations to stimulate a strong proliferative response (tenfold or greater increase in proliferation compared with concurrent vehicle controls). 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 acid (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 [3H]thymidine incorporation in situ. All the chemical allergens tested provoked robust proliferative responses, with the stimulation indices recorded at both test concentrations reflecting only small changes in activity compared with previously recorded data. Exposure to vehicle (4:1 acetone:olive oil, AOO) 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 secretion, with particularly vigorous production of cytokine being stimulated following exposure to oxazolone or hexyl cinnamic aldehyde. In contrast, application of chemical allergens was not generally associated with elevated IL-12 p40 secretion. Exposure of mice to PABA did not result in increased IFN-gamma or IL-12 production compared with vehicle-treated controls. In general, however, cytokine secretion did not correlate closely with the induction of LNC proliferation. These data indicate that expression by allergen-activated LNC of IFN-gamma or IL-12 does not provide a reliable or sufficiently sensitive endpoint for the LLNA compared with [3H]thymidine incorporation in situ.