Intracellular expression of cytokines and granzyme B in auricular lymph nodes draining skin exposed to irritants and sensitizers

The murine local lymph node assay (LLNA) has been extensively utilized to evaluate sensitizing chemicals. However, there have been some concerns that its use to discriminate between classes of chemicals is minimal. It is thus desirable to identify better or alternative immune endpoints with in LLNA itself. Here, we evaluated the protein and/or mRNA levels of cytokines and granzyme B (GzmB), a cytotoxic lymphocyte product, to discriminate between sensitizers and irritants and to characterize the chemical sensitizers when used as supplemental indicators in LLNA endpoints. For this, CBA/N mice were topically treated daily with a well-known chemical sensitizer such as a strong contact sensitizer 1-chloro-2,4-dinitrobenzene (DNCB), a skin contact sensitizer 2-phenyl-4-ethoxymethylene-5-oxazolone (OXA), and a skin or respiratory sensitizer toluene 2,4-diisocyanate (TDI), and the non-sensitizing irritants, croton oil (CRO) and nonanoic acid (NA), for 3 consecutive days. The protein and/or mRNA levels in auricular lymph nodes draining the ear skin were then analyzed by real-time RT-PCR and immunoassay.The sensitizers, but not the irritants, evoked pronounced interleukin (IL)-2, IL-3 and IL-4 or interferon (IFN)-γ. Significantly, different sensitizers evoked different cytokine patterns of IL-4 and IFN-γ, as DNCB strongly up-regulated both IFN-γ and IL-4, OXA up-regulated IFN-γ strongly but IL-4 weakly, and TDI up-regulated IL-4 strongly but IFN-γ weakly. The sensitizers also strongly up-regulated GzmB mRNA, while the irritants had a much weaker effect. Thus, these cytokines and GzmB mRNA may be useful as additional endpoints for discriminating between irritants and sensitizers or contact and respiratory sensitizers in the LLNA.