<p>Transition metals such as nickel (Ni), cobalt (Co), palladium (Pd), chromium (Cr) and gold (Au) are widely used as alloys in jewelry and biomaterials such as orthodontic and orthopaedic appliances. These metals also cause cell-mediated allergic contact dermatitis (ACD) reactions in a significant proportion of the population upon prolonged direct exposure. The immune mechanisms underlying the response to these metals are not yet well defined. In the studies described in this thesis we therefore investigated the profile of cytokine responses to various metal ions <i>in vitro</i> and the relationship with the ACD reaction <i>in vivo</i>. In the first study, we investigated the relationship between the profile and magnitude of Ni<sup>2+</sup>-induced cytokine responses <i>in vitro</i> and the degree of <i>in vivo</i> reactivity to Ni<sup>2+</sup>. PBMC from Ni<sup>2+</sup>-reactive (ACD) and non-reactive control subjects were cultured with or without NiCl<sub>2</sub>. The numbers of IL-4-, IL-5- and IL-13-producing cells and the concentrations of IFN-γ, IL-10 and IL-13 produced were analysed by ELISpot and ELISA respectively. Ni<sup>2+</sup> elicited a mixed Th1- and Th2-type cytokine profile in PBMC from ACD subjects with a positive correlation observed between the levels of the elicited cytokines and the degree of patch test reactivity. Hence, suggesting an involvement of both Th1- and Th2-type cytokines in ACD to Ni<sup>2+</sup> and a direct association between the magnitude of the Ni<sup>2+</sup>-induced cytokine response overall and the <i>in vivo</i> reactivity to Ni<sup>2+</sup>. The impact of the regulatory cytokine IL-10 on Ni<sup>2+</sup>-induced Th1- and Th2-type cytokine responses in human PBMC was investigated in the next study. PBMC from blood donors with a history of Ni<sup>2+</sup> reactivity and non-reactive control donors were stimulated with Ni<sup>2+</sup> <i>ex vivo</i> with or without addition of human recombinant IL-10 (rIL-10) or neutralising mAb to IL-10. Depletion/enrichment experiments were performed to phenotype the Ni<sup>2+</sup>-specific cytokine producing cells. Exogenous rIL-10 significantly down-regulated the production of all cytokines but with a more pronounced effect on IFN-γ. IL-10 neutralisation, on the other hand, enhanced the levels of Ni<sup>2+</sup>-induced IFN-γ only. Ni<sup>2+</sup>-specific cytokine-producing cells in PBMC were found to be predominantly CD4<sup>+</sup> T cells. Thus, IL-10 may play a regulatory role <i>in vivo</i> by counteracting the ACD reactions mediated by CD4<sup>+</sup> T cells producing Th1-type cytokines. In the third study, we investigated the relationship between <i>in vivo</i> patch test reactivity to a number of metals (Ni, Co, Pd, Cr and Au) included in the standard and/or dental patch test series and <i>in vitro</i> responses to the metals in question. PBMC from metal patch test positive and negative control subjects were stimulated with a panel of eight metal salts and cytokine responses analysed by ELISpot and/or ELISA. A mixed Th1- (IL-2 and/or IFN-γ) and Th2-type (IL-4 and/or IL-13) cytokine profile was observed in PBMC from most metal allergic subjects upon <i>in vitro</i> stimulation with the metal(s) to which the subject was patch test positive. Our data suggest that other metals included in the standard and dental patch test series, just like Ni2<sup>+</sup>, induce a mixed Th1- and Th2-type cytokine profile in PBMC from ACD subjects <i>in vitro</i>. We further developed a simplified ELISpot protocol utilising plates precoated with capture monoclonal antibodies (mAb) and subsequent detection in one step using enzyme-labelled mAb, for enumerating the frequency of allergen (Ni<sup>2+</sup>)-specific cytokine producing cells. This was compared with a regular ELISpot protocol, with an overnight incubation for capture mAb adsorbtion and detection with biotinylated mAb followed by enzyme-labelled streptavidin. PBMC from Ni<sup>2+</sup>-reactive and non-reactive subjects were incubated with or without NiCl<sub>2</sub> and the enumeration of cells producing IFN-γ, IL-4 or IL-13 by the two protocols were compared. PBMC from Ni<sup>2+</sup>-reactive subjects showed significantly higher Ni<sup>2+</sup>-induced IL-4 and IL-13 responses and the number of antigen-specific cytokine-producing cells determined by the two ELISpot protocols correlated well. In a nutshell, our data point to the potential use of <i>in vitro</i> cytokine assays as diagnostic tools in distinguishing ACD subjects sensitised to different metals and non-sensitised subjects.</p>
| Identifer | oai:union.ndltd.org:UPSALLA/oai:DiVA.org:su-717 |
| Date | January 2005 |
| Creators | Minang, Jacob |
| Publisher | Stockholm University, Wenner-Gren Institute for Experimental Biology, Stockholm : Wenner-Grens institut för experimentell biologi |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Doctoral thesis, comprehensive summary, text |
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