Targeting interleukin-6: all the way to treat autoimmune and inflammatory diseases.

Interleukin (IL)-6, a cytokine featuring redundancy and pleiotropic activity, contributes to host defense against acute environmental stress, while dysregulated persistent IL-6 production has been demonstrated to play a pathological role in various autoimmune and chronic inflammatory diseases. Targeting IL-6 is thus a rational approach to the treatment of these diseases. Indeed, clinical trials of tocilizumab, a humanized anti-IL-6 receptor antibody have verified its efficacy and tolerable safety for patients with rheumatoid arthritis, Castleman's disease and systemic juvenile idiopathic arthritis, resulting in approval of this innovative biologic for treatment of these diseases. Moreover, a considerable number of case reports and pilot studies of off-label use of tocilizumab point to the beneficial effects of tocilizumab for a variety of other phenotypically different autoimmune and chronic inflammatory diseases. Elucidation of the source of IL-6 and of mechanisms through which IL-6 production is dysregulated can thus be expected to lead to clarification of the pathogenesis of various diseases.

Introduction

Interleukin-6 (IL-6), initially designated as a B cell differentiation factor 1, is a representative cytokine featuring redundancy and pleiotropic activity 2-4. In the early phase of infectious inflammation, IL-6 is produced by monocytes and macrophages immediately after the stimulation of Toll-like receptors (TLRs) with distinct pathogen-associated molecular patterns (PAMPs) 5. In noninfectious inflammations, such as burn or traumatic injury, damage-associated molecular patterns (DAMPs) from damaged or dying cells stimulate TLRs to produce IL-6 6. This acute IL-6 expression plays a central role in host defense by stimulating various cell populations. When acting on hapatocytes, IL-6 strongly induces a broad spectrum of acute-phase proteins such as C-reactive protein (CRP), serum amyloid A (SAA), fibrinogen, hepcidin, haptoglobin, and antichymotrypsin, whereas it reduces albumin, cytochrome P 450, fibronectin, and transferrin 7, 8 (Figure .

CRP is a good biomarker of inflammation and is used as such in clinical laboratory tests. Its expression mainly depends on IL-6 9. If the free concentration of the anti-interleukin 6 receptor antibody, tocilizumab is maintained in serum at more than 1 μg/ml, CRP remains negative 10, so that the serum CRP level is a hallmark for checking whether IL-6 activity is completely blocked in vivo. Continuously high levels of hepcidin induced by IL-6 block iron transporter ferroportin 1 in macrophages, hepatocytes, and gut epithelial cells and lead to hypoferremia and anemia of chronic inflammation 11, whereas long-term high levels of SAA result in amyloid A amyloidosis 12. In lymphocytes, IL-6 induces B cell differentiation into immunoglobulin-producing cells 1. When CD4-positive naïve T cells are primed, a specific cytokine prompts their differentiation into an effector T cell subset. IL-6 together with TGF-β preferentially promotes differentiation of IL-17-producing T helper cells (Th17) that play a crucial role in the induction of autoimmune tissue injury, whereas IL-6 inhibits TGF-β-induced regulatory T cell (Treg) differentiation 13, 14. The resultant Th17/Treg imbalance leads to breakage of immunological tolerance and is of pathological importance for the development of various autoimmune and chronic inflammatory diseases 15. IL-6 also induces CD8-positive T cells to generate cytotoxic T cells 16. The function of IL-6 in hematopoiesis is to induce maturation of megakaryocytes into platelets as well as activation of hematopoietic stem cells 17. IL-6 production in bone marrow stromal cells generates the receptor activator of NF-kappaB ligand (RANKL), which is an essential factor for the differentiation and activation of osteoclasts and bone resorption, thus leading to osteoporosis 18. Enhanced angiogenesis and increased vascular permeability are pathological features of inflammation, and these characteristics are due to the excess production of vascular endothelial growth factor (VEGF), which is induced by IL-6 in inflamed lesions such as seen in synovium tissue of rheumatoid arthritis 19. The promotional activities of IL-6, such as the proliferation of keratinocytes or collagen production in dermal fibroblasts, may contribute to autoimmune skin diseases including psoriasis and systemic sclerosis 20, 21. Furthermore, IL-6 stimulates the growth of cells such as myeloma/plasmacytoma cells and mesangial cells 22-24.

IL-6 triggers signal transduction after binding to the IL-6 receptor (IL-6R) 25, 26. There are two forms of IL-6R, a transmembrane 80-kDa form with a short cytoplasmic domain and a soluble form (sIL-6R). After binding of IL-6 to transmembrane IL-6R, the resultant IL-6/IL-6R complex associates with gp130 27-29, and the activated IL-6 receptor complex is formed as a hexameric structure consisting of two molecules each of IL-6, IL-6R and gp130 (so-called a classical signaling) 30, 31. The expression of transmembrane IL-6R is limited to a few cell types but the IL-6/sIL-6R complex can also transduce the IL-6 signal to various cells which do not express transmembrane IL-6R but express gp130 (known as a trans-signaling mechanism) 32, so that IL-6 affects a wide variety of cells.

Pathological Role of IL-6 in Development of Diseases

When IL-6 is synthesized transiently, it promptly participates in the host defense against environmental stress such as infection and injury and at the same time provides an SOS (warning) signal by triggering a broad spectrum of biological events. Once the source of stress is removed from the host, IL-6-mediated activation of the signal transduction cascade is terminated by negatively regulatory systems in conjunction with the normalization of serum IL-6 and CRP levels. However, dysregulated persistent IL-6 production has been implicated in the development of various autoimmune, chronic inflammatory diseases and even cancers 2-4, 33. The reason(s) why such dysregulated continuous IL-6 production is induced remains to be clarified and elucidation of the mechanism(s) underlying persistent IL-6 synthesis in diseases is of particular importance to make their pathogenesis clear. It was found that in human immunodeficiency virus (HIV)-positive cases of multicentric Castleman's diseases all patients were infected with the Kaposi sarcoma-associated herpes virus (KSHV) and that sustained synthesis of both virus-derived IL-6, which directly binds to and stimulates human gp130, and of host-derived human IL-6 contribute to the development of the disease 34.

Moreover, numerous animal models of diseases have also disclosed the pathologic role of IL-6 in disease development and that IL-6 blockade by means of gene-knockout or administration of anti-IL-6 or anti-IL-6R antibody can suppress such disease development either preventively or therapeutically. For example, IL-6 blockade strategy demonstrably limited susceptibility to Castleman's disease-like symptoms in IL-6 transgenic mice 35, as well as in various mouse models of rheumatoid arthritis 36-48, systemic lupus erythematosus 49-51, scleroderma 52, 53, C-peptide-induced myositis 54, experimental autoimmune uveoretinitis 55, 56, experimental autoimmune encephalomyelitis 57, and many other diseases.

Targeting IL-6: All the Way to Treat Autoimmune and Inflammatory Diseases

Because of the pathological role of IL-6 in various diseases, blockade of IL-6 was expected to constitute a novel treatment strategy for these diseases 3, 58, 59. Consequently, a humanized anti-human IL-6R monoclonal antibody (chemical name: tocilizumab, generic name: Actemra outside of the EU or RoActemra inside the EU) was developed, by grafting the complementarity-determining regions of a mouse anti-human IL-6R antibody onto human IgG1. The resultant tocilizumab then blocks IL-6-mediated signal transduction by inhibiting IL-6 binding to transmembrane and soluble IL-6 receptors.

Clinical trials of tocilizumab have demonstrated its outstanding efficacy for rheumatoid arthritis 60-66, systemic juvenile idiopathic arthritis 67-71 and Castleman's disease 72, 73. For patients with moderately to severely active rheumatoid arthritis, tocilizumab is now being used as an innovative drug in more than 90 countries worldwide. As a monotherapy or in combination with disease-modifying antirheumatic drugs, it has significantly suppressed the disease activity and radiographically detected progression of joint deformity, thus improving daily functional activity. Tocilizumab was also approved as the first line biologic for the treatment of systemic juvenile idiopathic arthritis in Japan, India, USA and EU and for Castleman's disease in Japan and India.

Furthermore, favorable results of recent pilot studies, case series or case studies have suggested that tocilizumab may have broad application for other diseases. These diseases include systemic autoimmune diseases such as systemic lupus erythematosus 74-76, systemic sclerosis 77, polymyositis 78, vasculitis syndrome including giant cell arteritis 79-84, Takayasu arteritis 79, 82, 85-87, cryoglobulinemia 88, myeloperoxidase-antineutrophil cytoplasmic antibody-associated crescentic glomerulonephritis 89 and rheumatoid vasculitis 90. The application of tocilizumab may also extend to organ-specific autoimmune diseases including Crohn's disease 91, relapsing polychondritis 92, 93, acquired hemophilia A 94, autoimmune hemolytic anemia 95, 96, as well as to chronic inflammatory diseases such as adult-onset Still's disease 97-113, amyloid A amyloidosis 114-120, polymyalgia rheumatica 79, 84, 121, remitting seronegative symmetrical synovitis with pitting edema 122, Behcet's disease 123, 124, uveitis 125, graft-versus-host diseases 126, 127, and tumor necrosis factor receptor-associated periodic syndrome 128 (Table ). Some studies have reported that tocilizumab is efficacious for spondyloarthritis 129-135, although others observed only minor effects 136-138. In addition, tocilizumab is reportedly effective for pulmonary arterial hypertension 139-141, atopic dermatitis 142, and sciatica 143. Finally, it was observed that during tocilizumab treatment of patients with rheumatoid arthritis, HbA1c levels and insulin resistance indices such as the homeostasis model assessment of insulin resistance (HOMA-IR) and the leptin-to-adiponectin ratio improved 144, 145, while serum levels of reactive oxygen metabolites decreased 146. It can thus be expected that long-term tocilizumab treatment may offer protection against the progression of atherosclerosis leading to cardiovascular events 147. Indeed, large-scale genetic analyses demonstrated a causal association between IL-6R-related pathways and coronary heart disease 148, 149, and a randomized, open-label, parallel-group, multicenter study to evaluate the rate of cardiovascular events of tocilizumab in comparison to a TNF inhibitor, etanercept in patients with rheumatoid arthritis (ClinicalTrials.gov, Identifier: NCT01331837) is now in progress. To establish broad clinical indications for tocilizumab for various diseases, however, further clinical studies will be needed to verify its efficacy and safety. The current clinical trials are listed in Table .

On the basis of the pathologic role of IL-6 and the outstanding beneficial effect of tocilizumab, targeting IL-6 is a rational strategy for the treatment of various diseases and other biologics of IL-6 inhibitors are also being developed 32. These include fully human anti-IL-6R, anti-IL-6R nanobody, anti-IL-6 antibody, and anti-IL-6/anti-IgG avimer protein consisting of the IgG-binding domain fused to the N-terminus of a 3-domain IL-6 binding region, which results in a 19-kDa heterotetrameric avimer. These novel biologics block IL-6-mediated both classical and trans-signaling pathway by inhibiting IL-6 binding to both transmembrane and soluble IL-6R. By contrast, the fusion protein soluble gp130-Fc selectively targets IL-6/sIL-6R trans-signaling pathway. It is hypothesized that IL-6 trans-signaling is a local and temporal danger signal with fewer and less important physiological functions under non-stressed conditions than classical signaling on the basis of several animal models 32, 150.

Future Directions

IL-6 participates in the host defense against environmental pathogens, whereas dysregulation of IL-6 production has been implicated in the development of various autoimmune and chronic inflammatory diseases 2-4, 33. The pleiotropic activity of IL-6 also indicates that IL-6 blockade represents a rational treatment strategy for various diseases. A good example of the efficacy of such treatment is the dramatic improvement engendered by tocilizumab in amyloid A amyloidosis and anemia of inflammation through inhibition of their respective responsible proteins, SAA and hepcidin synthesis 151, 152. However, the mechanisms through which tocilizumab exerts its therapeutic effects on various phenotypically different autoimmune and inflammatory diseases are not yet well understood. In recent years, it has been shown that Th17 and/or Th1 >> Treg causes the onset of various autoimmune and chronic inflammatory diseases 14, 15. IL-6, in combination with TGF-β, promotes the differentiation of naïve T cells into Th17, but inhibits TGF-β-induced Treg differentiation, indicating that IL-6 is a very important factor for determining the Th17/Treg balance 14. Dysregulated IL-6 production leads to predominance of Th17 over Treg but anti-IL-6R antibody can repair this imbalance. It has been demonstrated in several animal disease models that IL-6 blocking suppresses antigen-specific Th17 and/or Th1 differentiation but induces antigen-specific Treg 47, 48, 55-57. Furthermore, it has been shown that tocilizumab in fact corrects Th17/Treg imbalance in rheumatoid arthritis patients 153. In another study, it was found that tocilizumab induced a significant reduction in the peripheral pre-switch and post-switch memory B cells of rheumatoid arthritis patients 154 and that tocilizumab but not the TNF inhibitor significantly reduced somatic hypermutation in immunoglobulin gene rearrangements in pre-switch memory B cells 155, thus suggesting that modulation of memory B cells may be one possible target for tocilizumab. Moreover, tocilizumab treatment led to a reduction in the pathologic CD38highCD19lowIgDnegative plasma cells of SLE patients 74 and could lessen the survival of plasmablasts, which produce the anti-aquaporin 4 antibody in neuromyelitis optica 156. These findings suggest that the clinical effect of tocilizumab is also mediated through its inhibition of pathological autoantibody production.

Because of the therapeutic efficacy of tocilizumab, IL-6 plays a major role in the onset or development of various phenotypically different diseases. IL-6 is produced by a panoply of cells including monocytes, macrophages, dendritic cells, T and B cells, neutrophils, mast cells, fibroblasts, synovial cells, keratinocytes, endothelial cells, stromal cells, mesangial cells, glial cells, neurons, chondrocytes, osteoblasts, smooth muscle cells, and others in response to various stimuli 2-4, 33. Such phenotypic difference of diseases is conceivably due to differences in cells which generate IL-6 through abnormal transcriptional activation of the IL-6 gene 157, 158 and/or inhibition of IL-6 mRNA degradation 159, 160, or dose-dependent effects of IL-6 produced by cells recruited into the organs. Some virus products from KSHV, human immunodeficiency virus (HIV), human lymphotropic virus-1 (HTLV-1) and hepatitis B virus have been reported to affect IL-6 gene activation and/or mRNA degradation 161-168. Therefore clarification of the cell source of IL-6 production and of the mechanism(s) through which dysregulated continuous IL-6 synthesis is induced constitutes an important issue for future studies into the pathogenesis of diseases.

Table 1

Application of IL-6 blockade strategy for various autoimmune and chronic inflammatory diseases.

Approved or candidate diseases		References
RA	> 90 countries worldwide*	60-66
Systemic JIA	Japan, India, USA and EU*	67-71
Castleman's disease	Japan and India*	72,73
SLE	Phase I, case reports	74-76
Systemic sclerosis	Case series	77
Polymyositis	Case series	78
Vasculitis syndrome	Case report & series	79-90
Crohn's disease	Pilot randomized trial	91
Relapsing polychondritis	Case reports	92,93
Acquired hemophilia A	Case report	94
Autoimmune hemolytic anemia	Case reports	95,96
Adult-onset Still's disease	Case reports & series	97-113
Amyloid A amyloidosis	Case reports	114-120
Polymyalgia rheumatica	Case reports & series	79,84,121
RS3PE	Case report	122
Behcet's disease	Case reports	123,124
Uveitis	Case report	125
GVHD	Case report & series	126,127
TRAPS	Case report	128
Spondyloarthritides	Case reports	129-135
Pulmonary arterial hypertension	Case reports	139-141
Atopic dermatitis	Case series	142
Sciatica	Case series	143

Tocilizumab, a humanized anti-IL-6 receptor antibody, has been approved* as a biological drug for the treatment of RA, Castleman's disease and systemic JIA, and is expected to be applicable to various other autoimmune and chronic inflammatory diseases. RA: rheumatoid arthritis; JIA: juvenile idiopathic arthritis; SLE: systemic lupus erythematosus; RS3PE: remitting seronegative, symmetrical synovitis with pitting edema; GVHD: graft-versus-host disease; TRAPS: tumor necrosis factor-associated periodic syndrome.

Table 2

Clinical trials of tocilizumab for diseases other than RA.

Targeted diseases	Identifier
ClinicalTrials.gov (USA)
Adult-onset Still's disease	NCT01002781
Relapsing polychondritis	NCT01041248
Type II diabetes, obesity	NCT01073826
Ankylosing spondylitis	NCT01209702
Graves' ophthalmopathy	NCT01297609
Cardiovascular disease in rheumatoid arthritis	NCT01331837
Polymyalgia rheumatica	NCT01396317
Giant cell arteritis	NCT01450137
Acute GVHD	NCT01475162
Non-ST elevation myocardial infarction	NCT01491074
Systemic sclerosis	NCT01532869
Transplant rates in highly sensitized patients awaiting kidney transplantation	NCT01594424
EU Clinical Trials Registry
Ankylosing spondylitis	2009-017488-40
	2009-017443-34
Cardiovascular disease in rheumatoid arthritis	2010-020065-24
Graves' ophthalmopathy	2010-023841-31
Systemic sclerosis	2011-001460-22
UMIN-CTR clinical trials (Japan)
ANCA-associated vasculitis	UMIN000002892
Systemic sclerosis	UMIN000005550
Neuromyelitis optica	UMIN000005889
	UMIN000007866
Chronic glomerulonephritis	UMIN000006080
Colorectal cancer	UMIN000007493
Takayasu arteritis	UMIN000007845

Current clinical trials of tocilizumab in the USA, EU and Japan are listed. GVHD: graft-versus-host disease; ANCA: anti-neutrophil cytoplasmic antibody.