Molecular Regulation of Immune Responses Provoked by Chemical Allergens

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Abstract

Allergic contact dermatitis or IgE mediated asthma and/or rhinitis can be provoked from exposure to the chemical allergens dinitrochlorobenzene (DNCB) or trimellitic anhydride (TMA), respectively. The regulation of these immune responses occurs mainly from specific subsets of T cell populations, such as T helper (Th) and T cytotoxic (Tc) cells. It is known that transcription factors and epigenetic regulation influences cell fate, and it is assumed that these molecular mechanisms are responsible in T cell development. A prolonged topical exposure regime of either DNCB or TMA in BALB/c mice leads to the preferential development of a Th1 or Th2 lymphocyte population, respectively. Assessment of this Th cell polarisation for this report was by the extra cellular secretion of cytokines; IFN-γ and IL-12 (Th1 specific cytokines) and IL-4, IL-5, IL-10 and IL-13 (Th2 specific cytokines) from allergen activated lymphocytes. The aims of this project are to measure transcriptional changes in genes associated in Th cell polarisation, in allergen activated lymph node tissue over a time course representing Th cell development in vivo. Data in this report shows method development of a RT-PCR technique that used in vitro treated splenocyte tissue. The effects of DNCB and TMA on the cellularity and cytokine secretion of allergen activated skin draining lymph node cells, are shown. Lymph node cells that had been activated with DNCB failed to secrete adequate levels of IFN-γ, however TMA activated cells did secrete significant levels of IL-4, IL-10 and IL-13 cytokine. Therefore TMA activated tissue was investigated further, a kinetic study designed to plot T cell development in vivo was performed. Results from this kinetic experiment showed IL-4 and IFN-γ mRNA expression levels increased over time, however mRNA levels for IL-4 were three fold higher than the mRNA levels of IFN-γ and significantly higher than the vehicle control. The mRNA expression levels measured in this kinetic study were performed on an individual animal basis and results showed no inter animal variation. There were no apparent changes in the mRNA expression of transcription factor genes investigated within these whole lymph node cell populations. Therefore a study into the cellular phenotype of auricular lymph nodes was performed. In comparison to naïve tissue TMA activated tissue had a 10% increase in B220+ cells. A time point from the kinetic study was repeated and CD4+ (Th) cells were isolated and CD4+ depleted cell populations (Tc, etc.) were also prepared, a sample of whole lymph node cells before isolation treatment was also arranged. The mRNA expression levels in the treatment groups showed that IL-4 and IFN-γ segregated, with IL-4 being more highly expressed in CD4+ cells and IFN-γ being more highly expressed in CD4+ depleted cell populations, when compared with vehicle treated control. The mRNA expression of the transcription factor T-Bet, which is associated in the activation of the ifn-γ gene, was shown to have higher levels of mRNA in CD4+ depleted cell populations in comparison to CD4+ enriched populations; the same pattern of mRNA segregation as IFN-γ. Results here corroborate with previous published work indicating an active Th2 cell population and an active Tc1 population is present following prolonged TMA exposure. Unfortunately significant changes in transcription factor mRNA was not detected in this experiment, however, this research does suggest that T cell isolation techniques provide the best platform for the assessment of molecular responses in allergen activated tissue and should be utilized for future studies.

Keyword(s)

Cytokine ; Th1 / Th2 cells; mRNA expression

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