Cytokine profiling of chemical allergens in mice: Measurement of message versus protein

Chemical respiratory allergy is an important occupational health problem, but there are currently available no validated methods for hazard identification. There has been interest for some time in the application of cytokine profiling for the characterization of chemical allergens. We have now examined whether these cytokine expression patterns are regulated at the level of mRNA and/or protein production. Mice (BALB/c strain) were exposed topically to the contact allergen 2,4-dinitrochlorobenzene (DNCB), or to the respiratory allergen trimellitic anhydride (TMA). Thirteen days after the initiation of exposure, a single cell suspension of draining (auricular) lymph node cells (LNC) was prepared. Cells were cultured for 24–120 h and supernatants analyzed for cytokine protein by cytokine bead array. In parallel experiments total RNA was prepared from freshly isolated or cultured cells and cytokine gene expression was analyzed by ribonuclease protection assay (RPA) or by real time reverse transcription-polymerase chain reaction (RT-PCR). DNCB-activated LNC secreted high levels of the type 1 cytokines interferon (IFN)-γ and IL-12 compared with TMA-stimulated LNC. The converse type 2 pattern was observed following treatment with TMA. Freshly isolated LNC from TMA-treated mice displayed a selective type 2 cytokine mRNA profile as measured by RPA. In contrast, RNA isolated from DNCB-activated LNC displayed a more mixed cytokine phenotype with relatively low levels of transcripts for both type 1 and type 2 cell products. When cytokine gene expression was measured by the more sensitive real time RT-PCR technique, more vigorous expression of IL-4 was recorded for TMA-activated LNC compared with DNCB-activated LNC but there was no evidence for elevated IFN-γ transcripts for the latter treatment. The observation that IFN-γ mRNA expression is not increased in DNCB-activated LNC despite robust secretion of this cytokine indicates that production is controlled mainly at the level of translation of previously transcribed mRNA or of protein secretion. Furthermore, the preferential cytokine profile recorded following TMA exposure was more clearly contrasting when measured at the level of protein secretion rather than at the level of mRNA expression. Experience to date suggests that the measurement of induced cytokine profiles shows promise for the hazard identification and characterization of chemical respiratory allergens, and that the end point best suited for this purpose is cytokine secretion rather than mRNA expression.