T-Helper 22 Cell Type Responses to Nickel in Contact Allergic Subjects Are Associated with T-Helper 1, T-Helper 2, and T-Helper 17 Cell Cytokine Profile Responses and Patch Test Reactivity

Abstract

Introduction: Contact allergy to nickel (Ni) is a delayed-type hypersensitivity reaction mediated by Ni-reactive T cells producing the hallmark cytokines of several T-helper cell (Th) populations including IFN-γ (Th1), IL-4, IL-5 and IL-13 (Th2), and IL-17A (Th17). IL-22-expressing CD4+ cells, which could be either Th17 co-expressing IL-22 or Th22, expressing IL-22 in the absence of IL-17A, have also been found in Ni-provoked skin of allergic subjects. It has been unclear if Ni-reactive T cells consist of distinct Th cell type populations or if they secrete a mix of Th cell hallmark cytokines. The aim herein was to assess if cellular cytokine responses to Ni, in ex vivo-stimulated peripheral blood mononuclear cells (PBMCs) from Ni-allergic subjects, include not only Th1, Th2, and Th17 but also Th22 hallmark cytokines and to define if the cytokines are produced by distinct cell populations representing different Th profiles.

Methods: PBMC from Ni-allergic subjects (n = 15) with different degrees of patch test reactivity and non-allergic controls (n = 5) were in vitro stimulated with Ni. Cytokine levels in PBMC supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA) (IFN-γ, IL-2, IL-3, IL-5, IL-6, IL-13, IL-17A, IL-22, and IL-31). FluoroSpot was used to assess if individual Ni-reactive cells produced single, or combinations of, cytokines representing different Th profiles. Cytokine combinations analyzed were IL-17A/IL-22/IFN-γ, IL-5/IL-17A/IFN-γ, IL-13/IL-22/IFN-γ, and IL-5/IL-13.

Results: IL-22 as well as all other cytokines measured by ELISA were induced by Ni at higher levels in PBMC from allergic versus non-allergic subjects, with higher levels being associated with stronger patch test reactivity. The levels of most Ni-induced cytokines were positively correlated with each other; IL-2 displayed the highest correlation with other cytokines and IL-6 the lowest. FluoroSpot analysis showed that Th signature cytokines, IFN-γ (Th1), IL-5 and IL-13 (Th2), IL-17A (Th17), and IL-22 (Th22), were almost exclusively produced by distinct cell populations.

Conclusion: Distinct Th cell populations, including Ni-reactive cells displaying Th1, Th2, Th17, and Th22 cytokine profiles, are all increased in PBMC from Ni-allergic subjects and positively associated with patch test reactivity. The relevance of these different Th profile populations for the up- or down-regulation of inflammatory reactions in the skin of Ni-allergic subjects remains to be clarified.

Keywords: Antigen specificity; Cytokines; Enzyme-linked immunosorbent assay; FluoroSpot; Nickel allergy; T-cell responses.

Fig. 1.

ELISA analysis of cytokine responses induced by NiCl₂ (Ni) and tetanus toxoid (TT) in peripheral blood mononuclear cells (PBMCs) from Ni-allergic and non-allergic subjects. IFN-γ, IL-2, IL-3, IL-5, IL-6, IL-13, IL-17A, IL-22, and IL-31 ELISAs were used to analyze PBMC supernatants from allergic and non-allergic subjects. Allergic subjects are divided into groups with +3, +2, and +1 patch test reactivity and Cntrl indicates the control group. PBMCs were cultured in the presence of Ni (a) or TT (b) or with medium only. The cytokine levels in medium only were in general below the detection limit of the ELISAs, or very low, and were subtracted from the antigen-induced cytokine levels shown. The box plots show the median (line in the box), min and max (vertical Ts), and 25th and 75th percentiles (box) for the respective group. Individual responses are shown as gray circles. Indicated above the boxes are statistically significant differences between groups (p < 0.05).

Fig. 2.

Associations between cytokine levels secreted by PBMC stimulated with either Ni (a) or TT (b). * The correlation between the levels of different cytokines, as determined by Spearman rank test, is shown as r_s values indicated by numbers in each box and p values indicated by the color of the box. White boxes indicate that there was no statistical significance (n.s.).

Fig. 3.

FluoroSpot analysis (a–h) of peripheral blood mononuclear cell (PBMC) secretion of single-, double-, and triple-cytokine combinations after stimulation with NiCl₂ (Ni) or Candida albicans extract. FluoroSpot assays with different cytokine combinations were used to analyze PBMC from allergic subjects. The responses shown are the mean of three subjects with +++ patch test reactivity. Indicated below each pie chart is the average total number of spots found in triplicate samples. Each pie chart shows the proportion of spots derived from cells secreting one, two, or three cytokines. The percentage of spots representing cells producing one single cytokine is indicated. Shown from the left are data from FluoroSpot assays simultaneously measuring IFN-γ/IL-5/IL-17A, IFN-γ/IL-17A/IL-22, IFN-γ/IL-13/IL-22, and IL-5/IL-13. Single, double-, and triple-cytokine-positive spots are shown with colors indicated below each pie chart. Unstimulated PBMC yielded low spot numbers (0–5 spots/cytokine and no double or triple spots) that were subtracted from the antigen-specific responses shown in the graphs.

Fig. 4.

Images of IFN-γ/IL-5/IL-17A FluoroSpot analysis of peripheral blood mononuclear cell (PBMC) responses to NiCl₂ (Ni). The assay was performed as described in Materials and Methods and Fig. 3. Shown are the results obtained with Ni-stimulated PBMC from one Ni-allergic subject. Only one well from a triplicate is shown. The three cytokines were detected with different fluorophores (488 [green] for IFN-γ; 550 [orange] for IL-5; 640 [red but changed here to blue for graphical reasons] for IL-17A) and were analyzed separately using wavelength-specific filters in the reader (the first three images from left). Spots positive for more than one cytokine were identified based on their co-localization in a computerized overlay analysis. An overlay image is shown to the right.