N. Suzuki, Department of Immunology and Medicine, St Marianna University School of Medicine, Sugao 2-16-1, Miyamae-ku, Kawasaki 216-8511, Japan. E-mail: email@example.com
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.
Behçet's disease (BD) is a systemic inflammatory disease with a recurrent nature, and T lymphocytes play an important role in its pathogenesis .
We have reported that interferon (IFN)-γ-producing lymphocytes were detected in erythema nodosum (EN) of patients with BD, while lymphocytes producing interleukin (IL)-4 were rarely detected . We have observed infiltration of CD4+ and CD8+ T cells in intestinal lesions of BD, along with the expression of mRNAs of proinflammatory and T helper type 1 (Th1) cytokines/chemokines . Several reports have indicated the dominance of Th1 cytokines in BD [4–7].
Recently, the Th1/Th2 paradigm was challenged by the discovery of various subsets of T helper cells. Th17 cells produce a number of proinflammatory cytokines, including IL-17, IL-17F, IL-21 and IL-22. Development of Th17 cells from naive T cells requires transforming growth factor (TGF)-β and IL-6 in mice. In human Th17 development IL-1β and IL-23 are required, but the role of TGF-β is controversial [8–13]. These cytokines induce the expression of RAR-related orphan receptor C (RORC), which is the master transcription factor of Th17 cells. An increase of IL-17 production in inflammatory bowel disease  and multiple sclerosis  suggests that Th17 cell dysregulation may be involved in the pathogenesis of certain human autoimmune diseases. Involvement of Th17 cells has been suggested in BD. Several researchers have described that mRNAs and proteins of Th17-related cytokines are increased in BD [16,17]. More recent studies of BD have shown simultaneous and excessive production of both Th1- and Th17-related cytokines in peripheral blood and cerebrospinal fluid [18–20].
In the past decade, numerous studies have demonstrated that Th17 cells can turn into IFN-γ-expressing T cells in mouse Th1 disease models, called ‘Th1-like cells’, ‘IFN-γ-expressing Th17 cells’ or ‘Th17/Th1 cells’. Transferred Th17 cells caused the autoinflammation in pancreas [21,22], eyes  and colon [24,25] after converting into IFN-γ+IL-17+CD4+ T cells. There is a possibility that frequencies of the cell population affected the severity of experimental autoimmune encephalomyelitis . However, the existence of in-vivo plasticity of Th17 cells in human autoimmune diseases is not established.
In this study we have investigated in detail Th17-related cytokine productions and expression of Th17-associated signalling molecules in BD.
Patients and methods
We studied 11 patients (five females and six males) with BD. Their mean age [± standard deviation (s.d.)] was 39·2 ± 9·2 years (range 25–56 years). Patients fulfilled the diagnostic criteria proposed by the International Study Group of BD . Sixteen age- and sex-matched normal control (NC) blood donors served as control subjects. None of the patients had been treated with intermediate–high-dose corticosteroid therapy (more than 10 mg prednisone/day) or colchicine therapy (more than 0·5 mg/day). We excluded those who had cyclosporin and other immunosuppressive agents from the patient group.
We studied specimens of erythema nodosum (EN) tissue from five BD patients (three females and two males), compared with three specimens of primary EN without any other systemic immune diseases (primary EN).
This study was conducted with the approval of the institutional review boards and was registered with the University Hospital Medical Information Network–Clinical Trials Registry (UMIN000003806). Informed consent was obtained from all the individuals prior to enrolment in the study.
Isolation and culture of memory and naive CD4+ T cells (Fig. 1)
CD4+CD45RO– T cells and CD4+CD45RO+ T cells were purified from peripheral blood mononuclear cells (PBMC) by magnetic cell sorting with a human naive CD4+ T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Memory CD4+ T cells were divided into CD4+CD45RO+CCR7– (effector memory) and CD4+CD45RO+CCR7+ (central memory) T cells with a human central memory CD4+ T cell isolation kit (Miltenyi Biotec) . The naive CD4+ T cells were then cultured in vitro as described below and memory cells were used directly for cytokine staining and mRNA purification.
In-vitro differentiation of naive CD4+ T cells
In our preliminary experiments, we determined the optimal culture conditions for inducing in-vitro differentiation of naive CD4+ T cells. Briefly, T cells were activated by plate-bound 10 µg/ml anti-CD3 (Dako, Glostrup, Denmark), 1 µg/ml anti-CD28 (Dako) and 20 ng/ml IL-2 (R&D Systems, Minneapolis, MN, USA) for 4 days in the presence of several cytokines and anti-cytokine antibodies mentioned below (first culture), and were then stimulated for more 7 days with anti-CD3, anti-CD28 and IL-2 (second culture) [8–11].
Naive CD4+ T cells in the first culture for inducing Th0 cells were supplemented further with 10 µg/ml anti-IL-4 (Becton Dickinson, Franklin Lakes, NJ, USA) and 10 µg/ml anti-IFN-γ (Becton Dickinson). Those for inducing Th1 cells were supplemented with anti-IL-4 and 10 ng/ml IL-12 (R&D Systems); those for inducing Th2 cells were supplemented with anti-IFN-γ and 10 ng/ml IL-4 (PeproTech, Rocky Hill, NJ, USA); and those for inducing Th17 cells were supplemented with anti-IL-4 and anti-IFN-γ plus 20 ng/ml IL-6 (R&D Systems), 10 ng/ml TGF-β (R&D Systems), 20 ng/ml IL-23 (R&D Systems), 10 ng/ml IL-1β (R&D Systems) and 10 ng/ml tumour necrosis factor (TNF)-α (R&D Systems).
Intracellular cytokine staining
The memory CD4+ T cells freshly separated from PBMC and the CD4+ T cells recovered from in-vitro culture of naive CD4+ T cells were analysed for intracellular cytokine staining using an intracellular cytokine staining kit (BD Biosciences, San Diego, CA, USA), according to the manufacture's protocol.
The cells were stimulated in the presence of 10 µg/ml phorbol 12-myristate 13-acetate (PMA), 1 µg/ml ionomycin and 10 µg/ml brefeldin (all from Sigma-Aldrich, St Louis, MO, USA) for 4 h. Cells were stained intracellularly for IFN-γ and IL-17 (both from R&D Systems).
Total RNA was isolated from cells with an RNeasy kit (Qiagen, Hilden, Germany). Complementary DNA was synthesized with TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA, USA) using random hexamers as primers, in accordance with the manufacturer's instructions. Eukaryotic 18S ribosomal RNA was used as an endogenous control. We studied six combinations of TaqMan primers and probes for the following Th17-related molecules from Applied Biosystems: IFN-γ, IL-17, IL-23R, TGF-β receptor type 1, mothers against DPP homologue 2 (Drosophila) (Smad2) and RORC. We selected these six primer pairs for the analysis of memory and naive T cells according to our preliminary study using PBMC of BD patients (manuscript in preparation). All samples were analysed in duplicate or triplicate with an ABI Prism 7300 Fast Real-Time PCR System (Applied Biosystems). Relative expression of each gene was calculated by the 2ddCt method, and was compared to that in normal control cells.
Immunofluorescence staining of skin specimens
We deparaffinized skin tissue specimens and retrieved antigens. Serial sections of the skin lesions were analysed immediately. Sections were incubated overnight with anti-IL-17 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-IFN-γ (R&D Systems), anti-CD4 (Dako) and anti-CD8 (Dako). Goat anti-mouse Alexa 488, goat anti-rabbit Alexa 594, donkey anti-rabbit Alexa 488 and donkey anti-goat Alexa 594 were used for secondary antibody (all from Invitrogen, Oslo, Norway). Their fluorescence was analysed with a LSM510 (Carl Zeiss, Jena, Germany) confocal laser scanning microscope.
Each value was expressed as mean ± standard error of the mean (s.e.m.). The Wilcoxon rank sum test was performed with jmp statistical software 7·0 (SAS, Cary, NC, USA) .
mRNA expression of helper T cell-related molecules in patients with BD
We did not observe any significant differences in gene expression analyses of memory CD4+ T cells.
Naive CD4+ T cells were purified and were cultured in vitro to induce T helper cell differentiation (1st culture, Fig. 1). IFN-γ gene expression was increased significantly in BD after culture for inducing Th1 cells (P < 0·05) (Fig. 2a). mRNA expressions of IL-17 and IL-23R were increased significantly after culture for inducing Th17 cells (P < 0·05). mRNA expression of RORC increased in BD regardless of the culture conditions, except in cultures for inducing Th2 cells. It is thus suggested that naive CD4+ T cells are prone to differentiate into Th17 cells in patients with BD.
Naive BD CD4+ T cells were cultured with several Th17-related cytokines in the first culture, and effects of the cytokines on IL-17 mRNA expression after the second culture were studied (Fig. 2b). Addition of IL-23 induced IL-17 mRNA expression in BD. In the presence of anti-IL-4, anti-IFN-γ, IL-6, TGF-β, IL-1β, TNF-α and IL-23 mRNA expression of IL-17 increased significantly in BD.
IFN-γ+IL-17+ cells are prevalent in the memory CD4+ T cell population in patients with BD
In (d) and (e) of Fig. 3a, representative staining patterns were depicted where IFN-γ+IL-17+ cells increased in BD effector memory CD4+ T cells which had been purified freshly from PBMC. Frequencies of IFN-γ+IL-17+ cells in effector (CCR7–) and central (CCR7+) memory CD4+ T cells from BD patients were significantly higher than those in NC (P < 0·01) (Fig. 3b).
Frequencies of IFN-γ+IL-17– cells were significantly lower in the same populations of CD4+ T cells of BD than in those of NC (P < 0·05) (Fig. 3b).
We attempted to observe effector memory IFN-γ+IL-17+CD4+ T cell frequencies twice in four BD patients and seven NC to determine consistency or fluctuation between the two time-points of IFN-γ+IL-17+CD4+ T cell frequencies. Average duration of observation was 9·5 months in BD and 7·0 months in NC. Effector memory IFN-γ+IL-17+CD4+ T cell frequencies were repeatedly low in NC (n = 9, first time: 8·9 ± 1·2%, second time: 10·5 ± 1·3%) and the relatively smaller s.e.m. suggested that their fluctuation was marginal in NC. In contrast, the cell frequencies were very high in BD patients (n = 4, first time: 16·5 ± 7·8%, second time: 17·5 ± 7·5%), and the larger s.e.m. suggested that cell numbers of effector memory IFN-γ+IL-17+CD4+ T cells fluctuated substantially in patients with BD.
Confocal microscopic analysis of skin lesions in BD
Histological examination revealed that T cells infiltrated perivascular sites of superficial and deep dermal layer in both BD erythema nodosum (BD–EN) and primary EN (data not shown). Using serial sections of BD–EN lesions, we found that IFN-γ+ IL-17+-producing cells were prevalent and at least some of them were CD4+ cells in BD–EN (Fig. 3c and Table 1).
Table 1. Histological analysis of skin lesions in patients with Behçet's disease (BD).
Histological T cell subtypes
Erythema nodosum of five BD patients (BD–EN) and primary EN lesions without any other systemic autoimmune diseases from three patients (primary EN) were examined histologically for cytokine production. We analysed serial sections of the skin lesions of the eight patients. We found that interferon (IFN)-γ+ interleukin (IL)-17+CD4+ T cells were prevalent in BD–EN compared with primary EN using serial sections of skin specimens.
CD4+ T cells/whole T cells (%)
IFN-γ+CD4+ T cells (%)
IL-17+CD4+ T cells (%)
CD8+ T cells/whole T cells (%)
IFN-γ+CD8+ T cells (%)
IL-17+CD8+ T cells (%)
IFN-γ+IL-17+ T cells (%)
Cytokines and microenvironments of T cells affect helper T cell differentiation. The combination of TGF-β and IL-6 plays an essential role in development of Th17 cells from naive T cells in mice. In human Th17 development IL-1β and IL-23 are required, but the role of TGF-β is controversial [8–13]. These cytokines induce the expression of RORC, which is the master transcription factor of Th17 cells. Th17 cells express CCR6 and migrate through epithelial cells with CCL20, a CCR6 ligand, on the inflammatory organs in a murine model . RORC expression is not stable in T cells and in-vivo plasticity of Th17 cells was ascertained in murine models [21–26]; this concept has been applied to human autoimmune diseases recently by a number of researchers [31–36].
IFN-γ+IL-17+CD4+ T cell clones, which expressed IL-23R and RORC mRNA, were established from the inflammatory bowel site in patients with Crohn's disease . Th17 cells in peripheral blood with psoriasis , multiple sclerosis [33,34] and in synovial fluid with juvenile idiopathic arthritis [35,36] were converted to IFN-γ+IL-17+CD4+ T cells in the presence of IL-23.
Underlying mechanisms involved in the pathological effects of IFN-γ+IL-17+CD4+ T cells remain largely unknown. IFN-γ+IL-17+CD4+ T cells have been postulated to be prevalent in the cell population inside local organs with human autoimmune diseases [31–35]. These results suggest that the cell frequency and migration ability are important to understand the role of IFN-γ+IL-17+CD4+ T cells in disease pathogenesis.
In BD, several researchers have described that Th17-related cytokines are increased in BD [16,17]. More recent studies showed simultaneous and excessive production of both Th1- and Th17-related cytokines in patients with BD [18–20]. Geri et al. reported that frequencies of both IFN-γ+CD4+ T cells and IL-17+CD4+ T cells were higher in the cerebrospinal fluid than those in the peripheral blood in BD .
In our study, the frequency of IFN-γ+IL-17+CD4+ T cells in peripheral blood of BD was increased significantly. IFN-γ+IL-17+ T cell numbers in BD–EN were higher than those in primary EN and at least some of them had CD4+ phenotypes. It is possible that BD IFN-γ+IL-17+CD4+ T cells share some common features with other human autoimmune diseases, and further investigation is necessary to prove this hypothesis.
Naive CD4+ T cells cultured in a Th17 cell-inducing condition expressed IL-23R mRNA excessively (Fig. 2a), and they exhibited enhanced IL-17 mRNA expression in the presence of IL-23 (Fig. 2b). IL-23R is expressed on memory T cells but not on naive T cells, and its expression is regulated by several cytokines, such as IL-6, IL-22 and IL-23 . Several investigators reported that IL-23 was essential not only to maintain but also to generate Th17 cells even in the absence of TGF-β[37,38]. We have become to hypothesize that IL-23 is involved in the immune aberration reported here.
Recent GWAS studies have identified IL-23R/IL-12RB genes as the disease-susceptible genes in BD [39,40]. Their studies suggest that both the Th17/IL-23 pathway and the Th1/IL-12 pathway may be dysregulated in patients with BD. Another study suggested that IL-23R gene conferred susceptibility to BD . Similarly, we found enhanced IL-23R mRNA expression in cultured naive CD4+ T cells in patients with BD. The relationship between IL-23/IL-23R and IFN-γ+IL-17+CD4+ T cell expansion needs to be studied in patients with BD.
In summary, IFN-γ+IL-17+CD4+ T cells increased in circulation and distribution over the skin lesions of BD. It is possible that they contribute to the pathophysiology of BD.
This work was supported in part by grants from Behçet's Disease Research Committee, Research on Specific Disease of the Health Science Research Grants from the Ministry of Health, Labor and Welfare, Japan.