Excessive CD4+ T cells co-expressing interleukin-17 and interferon-γ in patients with Behçet's disease

Abstract

Excessive T helper type 1 (Th1) cell activity has been reported in Behçet's disease (BD). Recently, association of Th17 cells with certain autoimmune diseases was reported, and we thus investigated circulating Th17 cells in BD. CD4(+) CD45RO(-) (naive) T cells were cultured with Th0-, Th1-, Th2- and Th17-related cytokines and antibodies, and their mRNA was studied by real-time polymerase chain reaction (PCR). When naive CD4(+) T cells were cultured with Th1- and Th17-related cytokines, interferon (IFN)-γ mRNA and interleukin (IL)-17 mRNA were up-regulated, respectively, in BD patients. Naive CD4(+) T cells cultured in a Th17 cell-inducing condition expressed IL-23 receptor (IL-23R) mRNA excessively. IL-17 mRNA expression was induced only when naive CD4(+) T cells were cultured in the presence of IL-23. CD4(+) T cells cultured with Th17 cytokines expressed excessive RAR-related orphan receptor C (RORC) mRNA. Using intracellular cytokine staining, we found that CD45RO(+) (memory) CD4(+) T cells producing IL-17 and IFN-γ simultaneously were increased significantly. Memory CD4(+) T cells producing IFN-γ but not IL-17 decreased profoundly in BD patients. CD4(+) T cells producing IL-17 and IFN-γ simultaneously were found in BD skin lesions. Collectively, we found excessive CD4(+) T cells producing IL-17 and IFN-γ (Th1/Th17) cells in patients with BD, and possible involvement of IL-23/IL-23R pathway for the appearance of excessive Th1/Th17 cells.

Fig. 1

Experimental protocol for cell preparation. Naive and memory CD4⁺ T cells were purified from peripheral blood mononuclear cells (PBMC) by magnetic cell sorting. The freshly separated memory CD4⁺ T cells were processed for intracellular cytokine analysis and mRNA purification. Naive CD4⁺ T cells were cultured with plate-bound 10 µg/ml anti-CD3, 1 µg/ml anti-CD28 and 20 µg/ml interleukin (IL)-2 for 4 days in the presence of several cytokines and anti-cytokine antibodies to induce directed differentiation of T helper cells. They were then stimulated for more 7 days with anti-CD3, anti-CD28 and IL-2.

Fig. 2

(a) Relative mRNA expression of T helper type 1 (Th1)- and Th17-related cytokines/receptors and signalling molecules in naive CD4⁺ T cells cultured in vitro[Behçet's disease (BD): n = 7, normal controls (NC): n = 7]. We treated naive CD4⁺ T cells in culture condition for inducing Th0 cells: 10 µg/ml anti-interleukin (IL)-4 and 10 µg/ml anti-interferon (IFN)-γ; for inducing Th1 cells: anti-IL-4 and 10 ng/ml IL-12; for inducing Th2 cells: anti-IFN-γ and 10 ng/ml IL-4; and for inducing Th17 cells: anti-IL-4 and anti-IFN-γ plus 20 ng/ml IL-6, 10 ng/ml transforming growth factor (TGF)-β, 20 ng/ml IL-23, 10 ng/ml IL-1β and 10 ng/ml tumour necrosis factor (TNF)-α. We selected six cytokines/receptors and signalling molecules from a panel of 25 cytokines/receptors and signalling molecules according to the results of preliminary gene expression analysis with peripheral blood mononuclear cells (PBMC). We did not observe any significant differences in gene expression analyses of memory CD4⁺ T cells between BD patients and NC. In the analysis of cultured naive CD4⁺ T cells, IFN-γ gene expression was increased significantly in BD patients in culture condition for inducing Th1 cells. mRNA expression of IL-17 and IL-23 receptors were increased significantly when cultured in the Th17 cell-inducing condition. mRNA expression of RAR-related orphan receptor C (RORC) was increased in BD patients regardless of the culture conditions except in cultures for inducing Th2 cells, suggesting that naive CD4⁺ T cells of BD patients were prone to differentiate into Th17 cells. Mean ± standard error of the mean (s.e.m.) is shown for each of seven BD and seven NC; *P < 0·05. (b) Relative mRNA expression of IL-17 in naive BD CD4⁺ T cells cultured with Th17-related cytokines. IL-17 mRNA was detected in the presence of IL-23 in BD patients. IL-17 mRNA was increased significantly in BD patients in the presence of Th17-related cytokines, especially when stimulated with IL-1β and TNF-α. aCD3: anti-CD3, aCD28: anti-CD28, aIFN-γ: anti-IFNγ, aIL-4: anti-IL-4, IL-23R: IL-23 receptor, TGF-βR1: TGF-β receptor type 1 and ND: not detected. Mean ± s.e.m. is shown in each of the seven BD and seven NC; *P < 0·05.

Fig. 3

Memory CD4⁺ T cells are shown by intracellular cytokine staining and also analysed with flow cytometry after phytohaemagglutinin (PHA)/ionomycin stimulation [Behçet's disease (BD): n = 8, normal controls (NC): n = 16]. (a) Effector (CCR7^–) memory CD4⁺ T cells were purified from BD patients and analysed with flow cytometry. Double staining was performed with (a) fluorescein isothiocyanate (FITC)-labelled isotype matched-control antibody and peridinin chlorophyll (PerCP)-labelled isotype-matched control antibody, (b) FITC-labelled isotype matched-control antibody and anti-human interleukin (IL)-17-PerCP, (c) anti-human interferon (IFN)-γ–FITC and PerCP-labelled isotype matched-control antibody, (d,e) anti-human IFN-γ-FITC and anti-human IL-17-PerCP. Panel (d) and (e) show frequencies of IFN-γ⁺ IL-17⁺, IFN-γ⁺IL-17^– and IFN-γ^– IL-17⁺ effector memory CD4⁺ T cells with NC and BD patients, respectively. The CD4⁺ T cells producing the cytokines simultaneously were detected in both BD patients and NC. Frequencies of IFN-γ⁺ IL-17⁺ CD4⁺ effector memory T cells were remarkably higher in BD patients than in NC. (b) Frequencies of IFN-γ⁺IL-17⁺ and IFN-γ⁺IL-17^– cells in each CD4⁺ T cell subpopulation with BD patients (dot-plots on right side in each square) and NC (left side). Frequencies of the IFN-γ⁺IL-17⁺ cells in effector memory (EM; CCR7^–) and central memory (CM; CCR7⁺) CD4⁺ T cells in patients with BD were significantly higher than those in NC (P < 0·01). Frequencies of IFN-γ⁺IL-17^– cells were significantly lower in the same populations of CD4⁺ T cells of BD patients than those of NC (P < 0·05). Box-plots with group means (bold blue lines) of eight BD and 16 NC are shown. (c) Confocal analysis of a skin lesion from BD patients. Double-colour staining on two consecutive slices of the skin lesions are shown. The upper three panels show staining with anti-CD4 (green) and anti-IL-17 (red). The lower three panels show staining with anti-IL-17 (green) and anti-IFN-γ (red). These images show that IFN-γ⁺IL-17⁺ cells appear in BD–EN and at least some of IFN-γ⁺IL-17⁺ cells are CD4⁺ (indicated by arrow).