Nickel-induced IL-10 down-regulates Th1- but not Th2-type cytokine responses to the contact allergen nickel

Abstract

Whereas the involvement of Th1- and Th2-type cytokines in contact allergy to nickel (Ni) is well documented, the role of the regulatory cytokine IL-10 is less clear. We therefore investigated the impact of IL-10 on Ni-induced Th1- (IFN-gamma) and Th2-type (IL-4 and IL-13) cytokine responses in human peripheral blood mononuclear cells (PBMC). PBMC from 15 blood donors with reactivity to Ni (Ni-PBMC) and 8 control donors devoid of reactivity (control PBMC) were stimulated with Ni and the frequency of cytokine-producing cells and the levels of secreted cytokines were analysed by ELISpot (IL-4, IL-13 and IFN-gamma) and ELISA (IL-10, IL-13 and IFN-gamma), respectively. The Ni-induced response was further assessed in the presence of recombinant IL-10 (rIL-10) or neutralizing antibody to IL-10 and the phenotype of the Ni-specific cytokine-producing cells regulated by IL-10 was determined by cell depletion experiments. Ni induced IL-10 production in Ni-PBMC (mean, (range); 33.1 pg/ml (0-93.4 pg/ml)) but not control PBMC (2.2 pg/ml (0-14.9 pg/ml)) (P = 0.002). Ni also induced significant production of IL-4, IL-13 and IFN-gamma that correlated with the IL-10 response. Addition of rIL-10 down-regulated the Ni-induced production of all cytokines but with a more pronounced effect on IFN-gamma. However, neutralization of Ni-induced IL-10 enhanced the levels of IFN-gamma induced by Ni (P = 0.004) but did not affect the number of IFN-gamma-producing cells or the production of other cytokines. Cell depletion experiments suggested that the Ni-specific IFN-gamma (and Th2-type cytokine) producing cells were CD4(+) T cells. The impact of IL-10 on Ni-induced IFN-gamma responses by CD4(+) T cells suggests that an important role of IL-10 in vivo is to counteract the allergic reactions mediated by Th1-type cytokines.

Fig. 1

Levels of IL-10 secreted by PBMC incubated in the absence or presence of 50 µM NiCl₂. The mean (range) of the spontaneous IL-10 levels were: for the control PBMC, 19 pg/ml (8·8–34 pg/ml) (n = 8; ○); Ni-PBMC, 19 (7·4–37 pg/ml) (n = 15; •). The statistical difference between the two groups following a Mann–Whitney U-test is depicted with asterisks (**P < 0·01) and is based on a comparison of the Ni-induced IL-10 levels minus the spontaneously produced IL-10 levels.

Fig. 2

Correlated levels of Ni-induced IL-10 and the Th1- or Th2-type cytokine production by PBMC. Plots depict correlation between levels of IL-10 and (a) the number of IL-4-producing cells or (b) the levels of IFN-γ. Plots are based on data after subtraction of spontaneous background. Values between 0 and 1 were set to 1. All Ni-PBMC and control PBMC are included in the figures (n = 23). The Ni-induced production of IL-13 measured by ELISpot and ELISA and IFN-γ measured by ELISpot correlated with IL-10 in a similar manner.

Fig. 3

Down regulation of Ni-induced cytokine production in Ni-PBMC by recombinant IL-10. PBMC were incubated with 50 µM NiCl₂ and a concentration range of rIL-10 followed by analysis of IL-13 and IFN-γ levels by (a) ELISA or (b) number of IL-4-, IL-13- and IFN-γ-producing cells by ELISpot. The effect of rIL-10 on the Ni-induced cytokine increment was calculated by subtracting the spontaneous cytokine production in the presence of rIL-10 from the Ni-stimulated cytokine production in the presence of the corresponding concentration of rIL-10. ○ (n = 8) and • (n = 7) represent Ni-reactive donors with levels of Ni-induced IL-10 < 30 pg/ml or > 30 pg/ml, respectively. Mean responses are indicated as horizontal bars. Statistical differences between the Ni-induced cytokine production in the absence of rIL-10 and after addition of different concentrations of rIL-10 following a Wilcoxon matched pairs test are depicted with asterisks (*P < 0·05, **P < 0·01, ***P < 0·001).

Fig. 4

Increased IFN-γ levels in response to Ni by neutralization of endogenous Ni-induced IL-10. PBMC were incubated with 50 µM NiCl₂ with or without α-hIL-10 mAb (10 µg/ml) and IL-13 and IFN-γ production were measured by (a) ELISA and (b) ELISpot. The Ni-induced cytokine increment was calculated by subtracting the spontaneous cytokine production in the absence or presence of α-hIL-10 mAb from the Ni-stimulated cytokine production at the same concentration of α-hIL-10 mAb. Control PBMC (n = 8) are shown in the left plot while Ni-PBMC (n = 15) are shown in the right plot; ○ (n = 8) and • (n = 7) represent Ni-PBMC with levels of Ni-induced IL-10 < 30 pg/ml or > 30 pg/ml, respectively. Mean responses at 0 or 10 µg/ml of α-hIL-10 mAb are indicated as horizontal bars. Statistical differences between the Ni-induced cytokine levels in the absence or presence of α-hIL-10 mAb following a Wilcoxon matched pairs test are indicated by asterisks (**P < 0·01).

Fig. 5

Phenotypic characterization of Ni-specific IL-4 and IFN-γ producing cells. Ni-PBMC were depleted of CD3⁺, CD4⁺ or CD8⁺ cells and (a, n = 6; c, n = 5) IL-4 and (b, n = 6; d, n = 4) IFN-γ production was determined by ELISpot (a, c) and ELISA (b, d). PBMC and depleted cell fractions were incubated at a concentration of 2·5 × 10⁶ cells/ml in the absence or presence of 50 µM NiCl_2. Cytokines produced spontaneously (□) and in response to Ni (▪) are depicted.