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Title: Cellular and chemical mechanisms of dendritic cell and T-cell activation by p-phenylenediamine
Author: Coulter, Eve M.
ISNI:       0000 0004 2670 4348
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2008
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The skin is one of the most common sites of exposure to chemicals. Some chemical entities are known to act specifically as skin allergens that can ultimately elicit the condition allergic contact dermatitis. This is defined as the presence of skin erythema and edema, resulting from a delayed-type antigen-specific T-cell-mediated reaction. One such chemical that has demonstrated immuno-stimulating capacity and is the focus of this thesis is p-phenylenediamine (PPD). PPD is widely used in the formulation of permanent hair dyes and is recQgnized as one of the most common chemical al1ergens. The sensitizing effects of PPD are believed to derive from the chemical's instability. Auto-oxidation in solution results in the formation of an electrophilic quinonediimine intermediate which is susceptible to sequential selfconjugation; the end product of these reactions is the trimer, Bandrowski's base (8B). Predictive animal models and patch testing have clearly identified PPD as a strong sensitizer; however, the role BB plays in the manifestation of allergic dermatitis remains uncertain. Thus, the aim of these studies was to investigate in detail, the chemical and cellular basis of PPD-mediated contact dermatitis. The purpose of initial experiments was to explore the relationship between PPD oxidation and functional maturation of human monocyte-derived dendritic cells in vitro. Exposure of dendritic cells to PPD (5-50 J,JM), but not BB, was associated with an increase in CD40 and MHC class II expression. These effects were not related to the direct toxic effects of PPD (neither apoptosis nor necrosis) or depletion of glutathione. PPD and 88 treatment did not alter basal cell surface expression of CD80, CD83, or CD86. Liquid chromatography-mass spectrometry (LC-MS) analysis revealed PPD was rapidly oxidized in cell culture media to 88. Addition of glutathione inhibited 88 formation but did not prevent covalent binding of PPD to dendritic cells protein or dendritic cell activation. To investigate in greater detail the chemical basis of PPD-mediated increased CD40 expression, using the same DC methodology, a range of structurally related disperse dyes were studied. Dendritic cell exposure to all disperse dyes was associated with an increase in CD40 expression; the rationale for this up regulation is attributed to their complex structures that are prone to auto-oxidation. PPD was also shown to directly stimulate proliferation of lymphocytes isolated from allergic patients, but not volunteers. In contrast, BB-specific T-cells were characterized from blood of allergic patients and volunteers, but not cord blood. Their presence seems to reflect an acquired immune response which is not translated into an allergic reaction. A comparison of the functional characteristics of T-cells from patients and volunteers using Luminex and micro-array technology identified an important difference in cytokine secretion and cytokine gene expression when patient and volunteer samples were compared. Specifically, stimulated cells from allergic patients secreted high levels and expressed high levels of cytokine genes associated with an inflammatory Th2 response (IL-4, IL-5, and IL-13). In contrast, cells from healthy individuals exemplified a more regulatory phenotype (IL-2ra/CD25, TGF-131 and IL-10). In conclusion, these experiments have characterized the degradation of PPD in solution and binding of PPD-derived material to cellular and extracellular protein, and how this relates to the activation of both dendritic cells and T-cells. Data showing the PPD-specific stimulation of Th2 secreting T-cells from allergic patients, but not volunteers, represents an important discrimination between allergic and non-allergic groups.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral