IL-6 was cloned as B cell stimulatory factor-2 (BSF-2) in 1986 (Hirano et al. 1986). This molecule has various biological activities; a strong stimulatory effect on growth of murine plasmacytoma and human myeloma, its functions as a hepatocyte stimulating factor, and the induction of acute phase reaction. IL-6 knockout (KO) mice revealed the inhibition of the antiviral antibody response after immunization with a vesicular stomatitis virus, compared with wild-type (WT) mice. Thus, IL-6 is a pleiotropic cytokine that is involved in the physiology of virtually every organ system.
Until recent years, it has been believed that Th1 cells mainly dominate the induction and progression of many autoimmune diseases. However, IFN-γ deficient (KO) mice do not show resistance to autoimmunity. On the contrary, they are even more susceptible to autoimmunity, which led us to hypothesize that there may be an additional Th subset that is distinct from Th1 cells. Recently, a new subset of Th cells that produces IL-17 (Th17) has been identified and was shown to have a crucial role in the induction of autoimmune diseases, such as rheumatoid arthritis and experimental autoimmune encephalomyelitis (EAE), and allergen-specific responses. The differentiation of Th17 cells from naïve T cells requires stimulation by IL-6 and TGF-β, which induces the master transcriptional factors of the Th17 subset such as retinoid-related orphan receptor γt (RORγt) and RORα (Fig. 1) (Korn et al. 2009).
IL-6 Receptors and Signaling
The IL-6 receptor (IL-6R) system consists of two polypeptide chains: an 80 kDa IL-6 receptor and a 130 kDa signal transducer (gp130) (Kishimoto 2005). IL-6R (80 kDa) exists in both a transmembrane form and a soluble form. IL-6 binds to both of these forms, which can then interact with gp130 and trigger signal transduction and gene expression. Gp130 is expressed ubiquitously in tissues. In addition to being a component of the IL-6R, gp130 is a component of receptors for other cytokines, such as leukemia inhibitory factor (LIF), IL-11, oncostatin M (OM), and cardiotropin-1 (CT-1), which explains the functional redundancy of IL-6 superfamily cytokines.
Negative Feedback Regulation of IL-6 Signaling
Although IL-6 is essential for the regulation of the immune process, overproduction of the cytokine causes inflammation and autoimmune diseases such as rheumatoid arthritis, systemic juvenile arthritis, and Crohn’s disease. Therefore, negative feedback regulation of IL-6 signaling is required for immune homeostasis. Cytokine signaling, such as IL-6 signaling, is negatively regulated by the suppressor of cytokine signaling (SOCS) and the protein inhibitor of activated STATs (PIAS). The SOCS family is composed of eight members: cytokine inducible SRC homology 2 (SH2)-domain-containing protein (CIS) and SOCS1 to SOCS7. SOCS-1, also called STAT-induced STAT inhibitor-1 (SSI-1) and JAK-binding protein (JAB), was initially identified as an intracellular negative-feedback molecule that inhibits JAK-STAT signaling initiated by various stimuli, including IFN-g, IL-4, IL-6, and leukemia inhibitory factor (LIF) (Krebs and Hilton 2000). SOCS-1 mainly inhibits IFN-γ signaling in vivo by binding to JAKs to inhibit its following signal transduction. However, SOCS-1 also negatively regulates innate immune responses such as lipopolysaccharide (LPS) -Toll-like receptor 4 (TLR4) signaling. Thus, SOCS-1 acts as an essential negative regulator in not only cytokine signaling but also TLR signaling.
Although SOCS-3 is also induced by cytokines such as IFN-γ and IL-6, and it can inhibit JAK activation as well as SOCS-1, SOCS-3 binds the cytokine receptor through its SH2 domain (Fig. 2). The activation of Stat1 and Stat3 induced by IL-6 is prolonged in SOCS-3-deficient tissues and cells, but not in SOCS-1-deficient tissues and cells, which indicates that SOCS-3 is a pivotal regulator of IL-6 signaling in vivo.
IL-6 and Immune Diseases
IL-6 is involved in many diseases such as rheumatoid arthritis, systemic-onset juvenile idiopathic arthritis, systemic lupus erythematosus, Crohn’s disease, and inflammatory bowel disease (Kishimoto 2005). In addition, IL-6 is involved in multiple sclerosis (MS), which is a chronic inflammatory disease affecting the central nervous system (CNS) white matter. Patients with MS exhibit higher mean levels of IL-6 in their cerebrospinal fluid than normal controls, and the treatment with an anti-IL-6 receptor monoclonal antibody (anti-IL-6R mAb) inhibited the development of EAE, which is a murine model of human MS that shares many pathological and histological characteristics with human MS.
IL-6 blockade seems to be an innovative treatment strategy for the numerous immune diseases that are impacted by IL-6 overproduction. Actually, blocking IL-6 signaling with a humanized anti IL-6R monoclonal antibody (tocilizumab) is an effective treatment for patients with autoimmune diseases such as Castleman’s disease and systemic onset juvenile arthritis (Venkiteshwaran 2009). Although many pro-inflammatory cytokines including IL-6, TNF-α, and IL-1β are increased in patients with autoimmune diseases such as rheumatoid arthritis and MS, the discovery of Th17 cells has defined IL-6 blockade as the potent and dominant treatment for these diseases. The details of the relationship between IL-6 and Th17 cells in autoimmune diseases will be discussed in section “IL-6 and Th17 Cells in Autoimmune Diseases”.
IL-17-Producing Helper T Cell (Th17)
Th17 cells produce IL-17A (IL-17), IL-17F, IL-22, IL-6, and TNF-α. The IL-17 family is composed of IL-17A, IL-17B, IL-17C, IL-17D, IL-17E (IL-25), and IL-17F. IL-17 is a potent inflammatory cytokine (Korn et al. 2009). As stated above, autoimmune diseases were previously assumed to be associated with dysregulated Th1 responses. However, IFN-γ deficiency did not attenuate some models of autoimmune diseases like EAE; on the contrary, IFN-γ deficiency worsened the disease. It was recently demonstrated that Th17 cells are dominantly associated with human and mouse autoimmune diseases such as rheumatoid arthritis, MS, and inflammatory bowel disease. In fact, IL-17 KO mice are resistant to the development of collagen-induced arthritis (CIA) and EAE, and IL-17 blockade by IL-17-blocking antibody prevents the development of EAE.
Th17 Cell Differentiation
Although initial reports claimed that IL-23 is required for the generation of Th17 cells from naïve T cells, it was subsequently demonstrated that IL-23R is not expressed on naïve T cells and that IL-23 acts as a survival signal for Th17 cells. At present, it is believed that Th17 cell differentiation is driven by the combination of IL-6 and TGF-β (Kimura and Kishimoto 2010). The orphan nuclear receptors, RORγt and RORα, are the key transcription factors that determine the differentiation of the Th17 lineage. IL-6 together with TGF-β induces these transcription factors, whereas IL-6 inhibits TGF-β-induced expression of Foxp3, a master transcriptional factor for Treg. The levels of RORγt and RORα are significantly reduced in Stat3-deficient T cells, but not in Stat1-deficient T cells under Th17-polarizing conditions, which indicates that Th17 cell differentiation is dependent on Stat3. In contrast to Stat3 activation, Stat1 activation inhibits the development of Th17 cells (discussed below). Although IL-6 activates both Stat3 and Stat1, Stat3 activation is maintained while Stat1 activation is suppressed in Th17 cells.
Conditioned medium from LPS-stimulated bone marrow-derived dendritic cells (DCCMs) can induce the production of IL-17 in naïve T cells. Interestingly, IL-17 was produced by DCCM even with the addition of anti-gp130 antibody or DCCM from IL-6 KO mice, which indicates that there is an IL-6-independent pathway in Th17 commitment. Although several cytokines including TNF-α and IL-23 participate in Th17 cell development, they are not required for the initiation of Th17 differentiation. What, then, is required for Th17 differentiation besides IL-6? It has been demonstrated that IL-21 acts as an initiator for Th17 commitment independently of IL-6. IL-21 is a novel cytokine produced by activated T cells and natural killer T (NKT) cells, and its receptor complex is composed of the common IL-2 receptor γ chain (γc) and IL-21 receptor (IL-21R). IL-21 and IL-6 inhibit TGF-β-induced Foxp3 expression and induce RORγt and RORα in a Stat3-dependent manner such that naïve T cells differentiate into Th17 cells.
Other Factors Involved in Th17 Cell Differentiation
The combination of IL-6 and TGF-β is unable to sustain the activation of Stat1 in Th17 cells, although it can sustain Stat3 activation. On the other hand, both Stat1 and Stat3 remained activated in Th17 cells induced by DCCM. These findings provide a novel and unknown basis for Th17 cell differentiation from naïve T cells. In fact, transcriptional factors such as Interferon-regulatory factor 4 (IRF4) and T-bet act as the positive and negative regulator for Th17 commitment, respectively. It has been also reported that retinoic acid inhibits Th17 cell development, and dioxin, a ligand of Aryl hydrocarbon receptor, promotes the generation of Th17 cells. Thus, IL-6 plays a central role in Th17 cell differentiation, whereas various factors regulate Th17 cell development.
Negative Regulation of Th17 Cell Differentiation
There is a negative regulatory system for Th17 cell differentiation. IL-27 and IFN-γ are responsible for the inhibition of its development in a Stat1-dependent manner. IL-27, another IL-12 family member, uses a receptor complex composed of IL-27R and gp130 to transduce its signal and activates both Stat1 and Stat3. Although both IL-6 and IL-27 transduce their signals via the gp130-JAK-STAT axis, IL-6 initiates Th17 commitment dependently on Stat3 activation, and IL-27 inhibits its development dependently on Stat1 activation. Additionally, IL-27 augments T-bet, the master transcriptional factor for Th1 cells, which indicates that IL-27 has both pro- and anti-inflammatory properties in Th cell differentiation.
It has been demonstrated that IL-2 also inhibits Th17 cell development. IL-2 cannot inhibit Th17 cell differentiation in Stat5-deficient T cells, and that there are Stat5 binding sites in the IL-17 promoter region; these findings suggest that Stat5 serves as a repressor. Thus, STAT family members activated by various cytokines provide positive and negative regulation of Th17 cell differentiation. However, the mechanisms of the regulation have not been elucidated.
Aryl Hydrocarbon Receptor: A New Player in Th17 Cells
Aryl Hydrocarbon Receptor
Aryl hydrocarbon receptor (Ahr), also known as dioxin receptor, is a ligand-activated transcription factor that belongs to the basic-helix-loop-helix-PER-ARNT-SIM family. Ahr is present in the cytoplasm, where it forms a complex with heat shock protein (HSP) 90, Ahr-interacting protein (AIP), and p23. Upon binding with a ligand, Ahr undergoes a conformational change, translocates to the nucleus, and dimerizes with Ahr nuclear translocator (Arnt). Within the nucleus, the Ahr/Arnt heterodimer binds to a specific sequence, the xenobiotic responsive element (XRE), which causes a variety of toxicological effects. Interestingly, it has been recently reported that Ahr is a ligand-dependent E3 ubiquitin ligase, which implies that Ahr has dual functions in controlling intracellular protein levels, serving both as a transcriptional factor to promote the induction of target proteins and as a ligand-dependent E3 ubiquitin ligase to regulate selective protein degradation. Ahr activated by ligands, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), regulates the generation of regulatory T cells (Tregs) and modulates the Th1/Th2 balance. In addition, it has been recently demonstrated that Ahr participates in Th17 cell differentiation. These data collectively demonstrate the importance of Ahr in the differentiation of T cell subsets.
Ahr Functions in Th17 Cells
Ahr is specifically induced in naïve T cells under Th17-polarizing conditions such as TGF-β plus IL-6 or TGF-β plus IL-21. Although the molecular mechanism of Ahr expression in Th17 development is not known, it is possible that its induction may be regulated downstream of Stat3 by IL-6 and TGF-β because Ahr expression was induced by TGF-β plus IL-6 in Stat1-deficient naïve T cells.
IL-6 and Th17 Cells in Autoimmune Diseases
The overproduction of IL-6 or abnormalities in its signal transduction is causative factors in autoimmune disorders including rheumatoid arthritis. At present, there is increasing clarification of the role of IL-6 in diseases such as RA in which Th17 cells are considered to be the primary cause of pathology. Humanized anti-IL-6 receptor antibodies (Tocilizumab) are currently being used clinically as an IL-6-blocking therapy for several autoimmune diseases. This section focuses on the relationship between IL-6 and Th17 cells in the pathogenesis of inflammatory diseases and an effective approach by Tocilizumab for the treatment of several autoimmune diseases.
IL-6 and Th17 Cells in Mouse Autoimmune Disease Models (CIA, EAE)
Previously, Th1 cells were considered to play a major role in pathogenesis of CIA and EAE. However, in IFN-γ-deficient mice and IFN-γ receptor-deficient mice, CIA and EAE symptoms are not ameliorated but rather exacerbated. In contrast, these diseases are suppressed in IL-17-deficient mice, and are alleviated by treatment with IL-17-neutralizing antibodies. These results indicate that in diseases such as CIA and EAE, Th17 cells are actually the major population in their pathogenesis and the in vivo differentiation and propagation of Th17 cells can be used as an index of these disease models.
Previous analyses revealed that IL-6 deficient mice are resistant to CIA and EAE. However, the reason for this resistance has been poorly understood. In recent years, it has been clarified that Th17 cells are induced from naïve CD4 T cells by TGF-β and IL-6 in vitro; therefore, it is conceivable that the impaired Th17 cell differentiation in these mice is the major cause for the resistance to CIA and EAE. However, in genetically deficient mice, their susceptibility to diseases such as CIA and EAE may be influenced by possible intrinsic defects in immune cells or nonuniform genetic backgrounds. Therefore, the administration of anti-IL-6 receptor antibodies to congenic wild-type mice with CIA or EAE is required to investigate the in vivo action of IL-6 in Th17 differentiation.
CIA is induced by administering type II collagen together with an adjuvant on Day 0 and Day 21 in mice. Interestingly, although anti-IL-6 receptor antibodies administered on Day 0 suppressed the induction of Th17 cells in the regional lymph nodes and the development of arthritis, antibodies administered on Day 14 did not suppress Th17 cells or arthritic symptoms. These results indicate that the inhibition of Th17 differentiation caused by anti-IL-6 receptor antibodies is necessary for CIA suppression and that for CIA in the in vivo environment; IL-6 is required for the initial differentiation from naïve T cells to Th17 cells, but not for the maintenance of Th17 cells after differentiation. Additionally, it was investigated whether a suppressing effect of Th17 cell development is observed with TNF inhibitor therapy in CIA. Intriguingly, when a TNF-soluble receptor (TNFR-Fc) was administered during the initial CIA induction period (Days 0–14), arthritis and Th17 differentiation could not be suppressed. However, when TNFR-Fc was administered after Day 21, arthritis is substantially suppressed without any effects on Th17 cell development. These results suggest that IL-6 inhibitor treatment in CIA acts primarily on initial CD4 T cell response including Th17 cell differentiation, rather than on the effector phase including angiogenesis and osteoclast differentiation. By contrast, it is suggested that the main point of action in TNF inhibitor therapy is different from that in IL-6 inhibitor therapy; it does not play a role in initial Th17 differentiation but it does act in the effector phase.
EAE is induced by administering the myelin sheath framework protein myelin oligodendrocyte glycoprotein (MOG) peptide together with an adjuvant and pertussis toxin. When anti-IL-6 receptor antibodies were administered immediately after antigen stimulation, the occurrence of EAE could be suppressed in the same manner as for CIA. In EAE models treated with anti-IL-6 receptor antibodies, no Th17 cells were found in draining lymph nodes or the spinal cord. Moreover, immune cells such as T cells, B cells, and macrophages were hardly observed in the lesion of spinal cord. On the other hand, the effect of anti-IL-6 receptor antibodies on Th17 cells and the disease onset was abolished when their administration was delayed. Thus, IL-6 is required for the initial differentiation phase for Th17 in the EAE model, and it appears that IL-6 also acts on cells other than Th17 cells. These results show that IL-6 inhibitor therapy is highly effective in suppressing the occurrence of EAE. Analyses of CIA and EAE indicate that initial Th17 differentiation phase in these autoimmune diseases is fundamentally dependent on IL-6, which suggests that IL-6 is a promising therapeutic target for autoimmune diseases involving Th17 cell inflammatory functions.
IL-6-Blocking Therapy in Human Autoimmune Diseases
In humans, the therapy by Tocilizumab has become a novel therapeutic strategy for some inflammatory and autoimmune diseases, including RA, systemic-onset juvenile idiopathic arthritis (JIA), Crohn’s disease (CD), Castleman’s disease, multiple myeloma, and systemic lupus erythematosus (SLE) (Venkiteshwaran 2009). Tocilizumab can block the IL-6 signals induced by the interaction of IL-6 and IL-6R. In the RA patients, Tocilizumab significantly improved the symptoms and ACR (American College of Rheumatology) improvement scores 20, 50, and 70 were 89%, 70%, and 47%, respectively, and normalized CRP and SAA in the patients within 6 weeks. Although a role of Th17 in RA is less clear, it has been reported that IL-17 is detected in the synovial fluid from RA patients and acts as a potent stimulator of osteoclastogenesis. Given that IL-6 is important for Th17 cell differentiation not only in mice but also in humans, Tocilizumab may improve the symptoms of RA through regulating the development of Th17 cells. As described above, it has been shown that IL-6 blockade by Tocilizumab is therapeutically effective for other inflammatory diseases such as Castleman’s disease, JIA, and CD; however, it is also still controversial whether Tocilitumab can inhibit Th17 cell differentiation in the improvement of these autoimmune diseases. It is required to demonstrate how Tocilizumab contributes to the treatment for above autoimmune diseases and whether Tocilizumab can also bring the therapeutic benefits for other autoimmune disorders.
In the past two decades, the knowledge on IL-6 has advanced from basic science to medicine. IL-6 is a pleiotropic cytokine that plays a major role in immune response, inflammation and hematopoiesis and its levels are increased in various autoimmune diseases. Tocilizumab is a humanized antihuman IL-6R antibody that inhibits the biological activities of IL-6 by blocking the binding of IL-6 to IL-6R. IL-6 blockade holds therapeutic value in autoimmune diseases, including Castleman’s disease, JIA, and RA. However, the precise reason why IL-6 blockade leads to the improvement of RA and other human autoimmune disorders is not well understood. The discovery of Th17 cells sheds light on the novel function of IL-6 and helps to address the above question. Th17 cells are IL-17-producing helper T cells and IL-17 is involved in the development of several autoimmune diseases. IL-6 is an essential factor for Th17 cell development, which is one of the reasons why targeting IL-6 activities is an effective approach. In the next phase, the potential of IL-6-targeting therapies in the treatment of various autoimmune diseases will be fully clarified.
We thank our colleagues in our laboratory for helpful discussion. This work was supported by the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation and Chugai-Roche Pharmaceutical Co. Ltd., Tokyo, Japan.