Allergic contact dermatitis (ACD) is a prevalent skin condition caused by chemical haptens, which enter the epidermis, and by modifying self-proteins, render them immunogenic via the activation of hapten-specific T-cells. It is currently not known which specific protein modifications are responsible for sensitization. This work investigates the extreme sensitizer DNCB, which confers a dinitrophenyl (DNP) protein modification. Immortalised keratinocytes (HaCaT), incubated with DNCB were compared to ex vivo human skin using immunofluorescence and western blot detection of DNP-proteins, showing widespread protein modification and similarities between tissue and cells when using a clinical dose. Proliferation assays using lymphocytes from DNCB-sensitive donors showed responses to DNP proteins isolated from DNCB-treated HaCaT cells and primary keratinocytes. While western blot analysis of pH gradient separated fractions identified a number of DNP-proteins in the DNP-HaCaT cell lysates, these were not immunogenic in lymphocyte assays. The model protein human serum albumin (HSA) was used to investigate the modification kinetics of DNCB by identifying which amino acid residues were changed more readily using a range of DNCB concentrations and incubation times. Cysteine residues, including those in disulphide bonds and particularly cysteine 34 are more readily modified than lysine in HSA, suggesting that DNCB is able to alter the structure of proteins. A novel process of hapten-reversal by a process termed ‘thiolysis’ was found to remove DNP groups from the cysteine residues of synthetic peptides derived from the sequence of HSA containing cysteine 34 using the reducing agent dithiothreitol. Identical peptides with C34>K34 showed no such hapten-reversal. This corresponds to the unexpected immunogenicity of the cysteinyl peptide and also the DNP modified tripeptide glutathione. Anti-DNP western blots show that the DNP group is transferred to other proteins during incubation with human monocytes in culture. This suggests a cellular process of removing DNP groups and GILT, a thiol reductase present in the endosomes is presented as a candidate for this process. This work demonstrates that DNCB can generate a wide variety of DNP protein adducts and that cysteinyl moieties are able to stimulate lymphocyte proliferation by way of hapten transfer. This highlights a potentially novel process involved in the mechanism of contact allergy.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:628781 |
| Date | January 2014 |
| Creators | Boyd, Peter |
| Contributors | Smyth, Neil ; Skipp, Paul |
| Publisher | University of Southampton |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://eprints.soton.ac.uk/369220/ |
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