CD8+ IL-17-producing T cells are important in effector functions for the elicitation of contact hypersensitivity responses

Abstract

Allergen-induced contact hypersensitivity (CHS) is a T cell-mediated delayed-type immune response which has been considered to be primarily mediated by CD8+ T cytotoxic type I (Tc1) cells. IFN-gamma, the prototype Tc1 (Th1) cytokine, has been implicated as the primary inflammatory cytokine for CHS. In this study, we demonstrate that neutralization of IL-17 rather than IFN-gamma suppresses the elicitation of CHS. The suppression does not result from inhibition of the proliferation of allergen-activated T cells. Allergen sensitization induces the development of distinct CD8+ T cell subpopulations that produce IFN-gamma or IL-17. Although CD8+ IL-17-producing cells are stimulated by IL-23, they are inhibited by IL-12, a prototypical stimulator of IFN-gamma-producing Tc1 cells. This indicates that CD8+ IL-17-producing cells are distinct from Tc1 cells and are important in effector functions at the elicitation of CHS. These studies provide insights into a novel mechanism for CHS.

Figure 1

Neutralization of IL-17 suppresses the elicitation of CHS. A). Sensitized mice were treated with anti-IL-17, anti-IFN-γ antibody or normal rat IgG prior to challenge. Naïve mice that were challenged served as negative controls. The difference between anti-IL-17 with anti-IFN-γ or rat IgG treated mice is significant at all time points (P<0.05). B). The expression of cytokines and chemokines in ear tissues was determined by RT-PCR. GAPDH served as a house keeping control. C). Relative expression level of each cytokine for each sample is calculated as density of cytokine/density of GAPDH. * undetectable.

Figure 2

Neutralization of IL-17 reduces edema and leukocyte infiltration in the skin. Ear skin samples were collected 24 hours after challenge and were subject to histology analysis and immunohistochemical staining. A). Histology of ear skin. Paraffin embedded sections were stained with H&E and pictures were taken microscopically with a 10x objective. The bar represents 50 μM. B). Immunohistochemical staining shows CD3+ T cells (red), CD11c+ monocytes/macrophages (green) and Gr-1+ granulocytes (green) in skin samples. The sections were counter-stained with fluorescence dye DAPI (blue). Pictures were taken microscopically with a 20x objective. C). Positive cells were counted in 10 fields of each group. The values are shown as mean ± SEM of 3 mice per group (* P<0.05; ** P<0.01). The data are representative of 2–3 independent experiments.

Figure 3

Production of IL-17 by hapten primed T cells. Primed T cells were placed in cultures with hapten labeled BM-DC for 48 hours and cytokines in supernatants were measured by ELISA. A). Hapten primed T cells produce IL-17 and IFN-γ. Treatment with LPS increased the ability of BM-DC (LPS) to stimulate IL-17 and IFN-γ production compared to untreated controls (Ctrl) (* P<0.05). Naïve T cells served as controls. The data (mean ± SD) are representative of 3 independent experiments. B). Effects of IL-12 and IL-23 on IL-17 production. CD4+ and CD8+ primed T cells were purified and cultured with hapten labeled BM-DC in the absence or presence of IL-12 or IL-23. The data (mean ± SD) are representative of three independent experiments. * P<0.05, compared to controls (None).

Figure 4

Characterization of hapten primed CD4+ and CD8+ T cells that produce IL-17 and IFN-γ. A). Cells were stained with anti-CD4 or CD8 (Alex488), IL-17 (PE) and IFN-γ (APC) antibodies. Control samples (Ctrl) were stained with PE and APC labeled isotype controls. The CD4+ or CD8+ T cells were gated and IL-17 or IFN-γ positive cells were analyzed. B). Cells were stained with ant-CD4 or CD8 (Alex488) and TCR beat chain antibody (APC). C). Cells were stained with ant-CD4 or CD8 (Alex488), IL-17 (PE) and TCR beat chain (APC) antibodies. Primed T cells that were stimulated with BM-DC without hapten labeling (Unlabeled DC) served as controls for those stimulated with hapten labeled DC (Labeled DC). CD4+ or CD8+ T cells were gated for analysis. The numbers indicate the percentage of IFN-γ or IL-17 positive cells in the gated CD4+ or CD8+ cell population. The data are representative of 3–4 independent experiments.

Figure 5

The role of IL-17 in CD8+ T cell mediated CHS. A). Mice were depleted of CD4+ (CD4del) or CD8+ (CD8del) T cells by in vivo injection of specific antibodies. The efficiency of depletion was determined by flow cytometry analysis of the draining lymph node cells. Numbers indicate percentages of positive cells. B). Mice were depleted of CD4+ (CD4del) or CD8+ (CD8del) T cells and then sensitized. CHS was measured following challenge. * P<0.05. C). Mice were depleted of CD4+ T cells and sensitized. The mice were then treated prior to challenge with anti-IL-17, IFN-γ antibody or control rat IgG. The treatment with anti-IL-17 antibody significantly reduces CHS at all time points (P<0.05). The data are shown as mean + SD of 3–4 mice per group and are representative of 2–3 independent experiments.

Figure 6

The role of IL-17 producing CD8+ T cells in transfer of CHS. Primed CD8+ T cells were purified by using MACS system and transferred intravenously into naïve Rag-1 deficient mice. The mice were treated with anti-IL-17 or rat IgG prior to challenge and CHS was measured. As a control (Ctrl), Rag-1 deficient mice were sensitized and challenged. Mice that were neither transferred with cells nor sensitized were challenged and served as naive control (Naïve). The difference between rat IgG and anti-IL-17 antibody treated group is significant at all indicated time points (P<0.05). The data are shown as mean ± SD of 3 mice per group and are representative of 2 independent experiments.

Figure 7

Neutralization of IL-17 does not affect primed T cells. A) Neutralization of IL-17 has no effect on the proliferation of primed T cells. Primed CD4+ or CD8+ T cells were purified and cultured with hapten labeled BM-DC in the presence of anti-IL-17 antibody or rat IgG. The data (mean ± SD) are representative of three independent experiments. B) Neutralization of IL-17 does not affect CHS following re-challenge (details in Materials and Methods). There is no significant difference between all treated groups of mice following re-challenge. The data (mean ± SD) are representative of 2 independent experiments.