Th17 differentiation

Naïve T cells stimulated in the presence of IL-12 become Th1 cells and express the transcription factor T-bet while Th2 cells express the transcription factor GATA-3. Initial studies in mice suggested that IL-23, a heterodimeric cytokine that shares a subunit with IL-12, induced IL-17 expression. However, subsequent studies demonstrated that the IL-23 receptor is only expressed on T cells after activation, and therefore IL-23 can up-regulate IL-17 in memory T cells but cannot act on naïve T cells to induce Th17 differentiation. Instead, the key to Th17 differentiation in the mouse is the combination of transforming growth factor (TGF)-β and IL-6. Tumor necrosis factor (TNF)-α and IL-1β can further enhance mouse Th17 differentiation, but only in the presence of TGF-β and IL-6.

Whereas Th1 and Th2 cells follow similar rules in humans as in mice, Th17 differentiation may not be as conserved, and IL-1β, IL-6, and IL-23 all appear to be important. Several studies suggest that TGF-β, a crucial cytokine for mouse Th17 differentiation, actually inhibits human Th17 development. Notwithstanding these claims, TGF-β likely is an inducer of human Th17 cells, but the effects of TGF-β are extremely concentration dependent; low doses induce Th17 differentiation, while high doses inhibit Th17 development and induce Tregs. T cells from cord blood, which represent a more naïve state than those in adult peripheral blood, do in fact require TGF-β to differentiate into Th17 cells. Additional recent studies also support a role for TGF-β in human Th17 differentiation.

IL-12, IFN-γ, and IL-4 can inhibit Th17 differentiation in both mouse and human. IL-17, on the other hand, does not appear to inhibit Th1 or Th2 differentiation, or does so very weakly, and so Th1 and Th2 cells typically dominate over Th17. This domination explains one of the ways in which TGF-β can promote Th17 differentiation—by suppressing production of the inhibitory cytokines IFN-γ and IL-4—but TGF-β also has direct roles in Th17 differentiation because it is required even in the absence of IFN-γ and IL-4. TGF-β synergizes with IL-6 to induce expression of the transcription factor RORγt, a key regulator of Th17 differentiation. In mice RORγt is both necessary and sufficient for IL-17 expression in vitro and in vivo, although its induction and action may be facilitated by a distinct transcription factor, Runx1. In humans RORC (the human orthologue of RORγt) is induced by IL-1β, IL-6 and IL-23, the same cytokines that induce IL-17. In addition, RORC expression is restricted to IL-17–producing clones.

In addition to RORγt, Th17 development in mice depends on the transcription factor STAT3, which is activated by IL-6 and IL-23. STAT3 has multiple roles in Th17 development: in activated Th17 cells stimulated with IL-23 it binds directly to the IL-17 promoter and induces IL-17 expression, and in naïve T cells stimulated with TGF-β and IL-6 it is required for induction of RORγt expression. IL-23 also activates STAT4, which is the primary mediator of IL-12 signaling and is required for Th1 differentiation, yet is still important for IL-23–induced IL-17 production. Thus STAT4 may inhibit Th17 development downstream of IL-12, while also supporting IL-17 expression downstream of IL-23.

Cytokines expressed by Th17 cells

Mouse Th17 cells specifically express IL-21 soon after activation, and autocrine IL-21 plays an important role in RORγt and IL-17 expression. IL-21 can also partially replace IL-6 during Th17 differentiation, giving established Th17 cells the ability to promote further Th17 development in neighboring cells. IL-23 in combination with TGF-β can also induce RORγt and IL-17 expression, but only after IL-6 or IL-21 induce IL-23 receptor expression. Thus IL-6, IL-21, and IL-23 act sequentially: first IL-6 up-regulates IL-21, then both IL-6 and IL-21 up-regulate IL-23 receptor, and finally IL-23 appears to up-regulate effector function and pathogenicity in Th17 cells. Recent evidence points to a role for IL-21 in human Th17 differentiation, and human IL-21 can also counteract suppression by Tregs.

IL-17, or IL-17A, is one member of a family of 6 cytokines known as IL-17A through F. Th17 cells specifically express IL-17F in addition to IL-17A. IL-17A and IL-17F are closely related, with 55% amino acid identity as well as a common receptor. IL-17A and IL-17F are both homodimeric cytokines, but human and mouse T cells also produce an IL-17A/F heterodimer that has potent inflammatory effects. It may be important to measure, as well as to target, both IL-17A and IL-17F in disease. Other proinflammatory cytokines produced by both mouse and human Th17 cells include TNF-α and IL-22. IL-22 induces antimicrobial proteins, defensins, acute-phase proteins, inflammatory cytokines, chemokines, and hyperplasia. However, IL-22 protects hepatocytes during acute liver inflammation ; thus IL-22 may either enhance inflammation or limit tissue damage induced by IL-17, depending on the type of tissue.

Unexpectedly, a subset of Th17 cells coexpresses IFN-γ, particularly in humans, where as many as half of all the IL-17 positive cells also express IFN-γ. These double positive cells complicate the idea that Th17 cells are a unique subset distinct from Th1 cells, and are particularly hard to explain given that IFN-γ has been shown to inhibit IL-17 expression. It is not clear yet whether these cells represent a stable phenotype, or a transitional phase from Th17 to Th1 or vice versa. Although there are no data on the specific role of these double positive cells, both IFN-γ and IL-17 are important mediators of inflammation, and cells that produce both cytokines are likely to contribute to pathogenesis in certain environments. Also unexpected is that Th17 cells coexpress IL-10, an anti-inflammatory cytokine. T-cell sources of IL-10 include Th2 cells and various types of Tregs, but Th1 cells also secrete IL-10 in certain conditions. In mice the combination of TGF-β and IL-6, which synergize to induce IL-17 production, also synergize to induce IL-10, such that half of the IL-17 positive cells coexpress IL-10. IL-10 produced by mouse Th17 cells may serve an important protective function by limiting inflammation and tissue damage normally caused by IL-17, through antagonistic effects on target tissues. It is not yet known whether human Th17 cells ever coexpress IL-10.

Cytokines expressed by Th17 cells

Mouse Th17 cells specifically express IL-21 soon after activation, and autocrine IL-21 plays an important role in RORγt and IL-17 expression. IL-21 can also partially replace IL-6 during Th17 differentiation, giving established Th17 cells the ability to promote further Th17 development in neighboring cells. IL-23 in combination with TGF-β can also induce RORγt and IL-17 expression, but only after IL-6 or IL-21 induce IL-23 receptor expression. Thus IL-6, IL-21, and IL-23 act sequentially: first IL-6 up-regulates IL-21, then both IL-6 and IL-21 up-regulate IL-23 receptor, and finally IL-23 appears to up-regulate effector function and pathogenicity in Th17 cells. Recent evidence points to a role for IL-21 in human Th17 differentiation, and human IL-21 can also counteract suppression by Tregs.

IL-17, or IL-17A, is one member of a family of 6 cytokines known as IL-17A through F. Th17 cells specifically express IL-17F in addition to IL-17A. IL-17A and IL-17F are closely related, with 55% amino acid identity as well as a common receptor. IL-17A and IL-17F are both homodimeric cytokines, but human and mouse T cells also produce an IL-17A/F heterodimer that has potent inflammatory effects. It may be important to measure, as well as to target, both IL-17A and IL-17F in disease. Other proinflammatory cytokines produced by both mouse and human Th17 cells include TNF-α and IL-22. IL-22 induces antimicrobial proteins, defensins, acute-phase proteins, inflammatory cytokines, chemokines, and hyperplasia. However, IL-22 protects hepatocytes during acute liver inflammation ; thus IL-22 may either enhance inflammation or limit tissue damage induced by IL-17, depending on the type of tissue.

Unexpectedly, a subset of Th17 cells coexpresses IFN-γ, particularly in humans, where as many as half of all the IL-17 positive cells also express IFN-γ. These double positive cells complicate the idea that Th17 cells are a unique subset distinct from Th1 cells, and are particularly hard to explain given that IFN-γ has been shown to inhibit IL-17 expression. It is not clear yet whether these cells represent a stable phenotype, or a transitional phase from Th17 to Th1 or vice versa. Although there are no data on the specific role of these double positive cells, both IFN-γ and IL-17 are important mediators of inflammation, and cells that produce both cytokines are likely to contribute to pathogenesis in certain environments. Also unexpected is that Th17 cells coexpress IL-10, an anti-inflammatory cytokine. T-cell sources of IL-10 include Th2 cells and various types of Tregs, but Th1 cells also secrete IL-10 in certain conditions. In mice the combination of TGF-β and IL-6, which synergize to induce IL-17 production, also synergize to induce IL-10, such that half of the IL-17 positive cells coexpress IL-10. IL-10 produced by mouse Th17 cells may serve an important protective function by limiting inflammation and tissue damage normally caused by IL-17, through antagonistic effects on target tissues. It is not yet known whether human Th17 cells ever coexpress IL-10.

Trafficking of Th17 cells

Human and mouse Th17 cells express the chemokine receptor CCR6, including memory cells from healthy peripheral blood and inflamed tissue, as well as in vitro primed naïve T cells. Although not all CCR6-positive cells are Th17, within the CCR6-positive population, those that coexpress CXCR3 are either Th1 or IFNγ-IL-17 double positive, whereas those that coexpress CCR4 secrete only IL-17. The majority of RORC expression is restricted to the CCR6+CCR4+ population, with a small amount in the CCR6+CXCR3+ population. CCR6 mediates homing to skin and mucosal tissues and is important in recruitment of pathogenic T cells in many inflammatory diseases now associated with IL-17, including psoriasis, inflammatory bowel disease, allergic asthma, and RA. Of note, the CCR6 ligand CCL-20 is expressed by Th17 cells and is up-regulated in stromal cells by IL-17, allowing Th17 cells in inflamed tissues to attract additional Th17 and Th1 cells. Another group found human memory Th17 cells within the CCR2+CCR5− population and Th1 cells within the CCR2+CCR5+ population. It is not yet clear whether the CCR6+CCR4+ population overlaps with the CCR2+CCR5−, but the combination of all 4 markers may be useful for isolating human Th17 cells from blood and sites of inflammation.

Role of Th subsets in collagen-induced arthritis and other RA models

RA is traditionally classified as a Th1-mediated disease, but mice treated with neutralizing antibodies to IFN-γ and IFN-γ receptor deficient mice develop more severe arthritis, and IFN-γ deficiency renders resistant strains of mice susceptible to collagen-induced arthritis (CIA). Further insight arises from the discovery that the IL-12 p40 subunit is shared by IL-23. IL-23 p19 deficient mice do not develop CIA, while IL-12 p35 deficient mice actually develop more severe disease. Both IL-12 and IFN-γ are potent suppressors of Th17 differentiation in vitro, suggesting a mechanism for the protective function of these cytokines. In vivo, arthritic mice deficient in IFN-γ or treated with neutralizing antibody to IFN-γ have elevated IL-17 in serum and in cultures of collagen restimulated spleens and lymph nodes.

The importance of IL-17 in the pathogenesis of arthritis has been shown in a variety of animal models, both genetic and adaptive. IL-17 knockout mice develop significantly less arthritis, and treatment with neutralizing antibodies to IL-17 or soluble IL-17 receptor alleviates joint inflammation. IL-17 receptor signaling in radiation-resistant cells of the joint is required for the induction of chronic destructive synovitis, cartilage damage, bone erosion, and inflammatory cytokine expression in acute streptococcal cell wall arthritis. In rat adjuvant-induced arthritis, IL-17 is important for both inflammation and joint destruction. In TNF-deficient mice, IL-17 can drive cartilage and bone destruction. IL-17 is also required for the development of spontaneous arthritis in other mouse models of RA, such as IL-1Ra deficient mice and SKG mice. IFN-γ plays a protective role in CIA by inhibiting Th17 cell development, but it may still have pathogenic effects in some instances. For example, the effect of neutralizing IFN-γ in CIA varies depending on the timing of administration, demonstrating that IFN-γ may be pathogenic in the early phases of CIA but protective in the later stages. In addition, the mouse model proteoglycan-induced arthritis is dependent on IFN-γ. Similarly, there may be subsets of RA patients with Th1- or Th17-dominant disease.

The role of IL-17 in RA

IL-17 is increased in RA sera and synovial fluid, and is present in the T-cell rich areas of the synovium. RA serum and synovial fluid contains much more IL-17 than osteoarthritis serum or synovial fluid. T cells cultured from normal donors or RA patients produce IL-17 in vitro following activation. A specific response of RA T cells to citrullinated but not uncitrullinated aggrecan is accompanied by robust secretion of IL-17. Increased levels of IL-17 and TNF-α mRNA expression in early RA synovium are predictive of more severe joint damage progression, whereas high levels of IFN-γ mRNA are predictive of protection from damage progression.

Further support for the role of Th17 cells in RA comes from in vitro studies showing the robust and widespread inflammatory effects of IL-17 on cells of the joint, IL-17 induces the production of inflammatory cytokines such as IL-1β, TNF-α, IL-6, and IL-23 by synovial fibroblasts, monocytes, and macrophages, all of which promote inflammation and Th17 development. IL-17 also induces an array of chemokines, including CXCL-1, CXCL-2, CXCL-5, CXCL-8, CCL-2, and CCL-20, leading to recruitment of T cells, B cells, monocytes, and neutrophils. Moreover, IL-17 directly stimulates monocyte migration. Leukocyte recruitment is further enhanced by up-regulation of granulocyte-colony stimulating factor and granulocyte macrophage-colony stimulating factor, as well as vascular endothelial growth factor (VEGF), an inducer of angiogenesis, and other angiogenic factors. IL-17 has been reported to induce production of proangiogenic factors from synovial fibroblasts, and to synergize with TNF-α in this effect. IL-17 can also orchestrate bone and cartilage damage. matrix metalloproteinases, nitric oxide, and RANK/RANKL, as well as inflammatory cytokines and chemokines, are up-regulated in chondrocytes and osteoblasts by IL-17. Th17 cells can also induce osteoclastogenesis directly by expressing RANKL (receptor activator of nuclear factor κB ligand) on their cell surface. SDF-1, which is abundant in RA synovium and promotes recruitment of CD4+ memory T cells, monocytes, and B cells, is induced by IL-17, although IL-1β and TNF-α are not efficient in inducing this factor.

TNF blockade does lower serum IL-17 levels, but IL-17 can induce inflammation and enhance cartilage and bone erosion in mice even under TNF and IL-1 neutralizing conditions. Combined TNF, IL-1β, and IL-17 blockade, was more effective at controlling IL-6 production and collagen degradation than was blocking TNF-α alone, in cultures of RA synovium. Similarly, combination blockade of TNF-α and IL-17 suppressed ongoing CIA and was more effective than neutralization of TNF-α alone. These results suggest that treatments designed to block IL-17 could be beneficial in RA.

Th17 cells produce IL-21, which acts to further enhance Th17 development and plays a role in CIA and RA. IL-21 is increased in RA serum and synovial fluid. Its receptor is highly expressed on peripheral blood and synovial fluid lymphocytes, as well as on synovial fibroblasts and macrophages from patients with RA. Administration of IL-21R fusion protein significantly reduced disease in CIA and rat antigen-induced arthritis.

IL-23, which enhances IL-17 production and Th17 effector function in humans and mice, is also implicated in arthritis. Mice deficient in the IL-23 p19 subunit develop less severe CIA. In RA patients IL-23 is elevated in sera and synovial fluid, with increased expression of IL-23 p19 subunit in synovial fibroblasts. Recently STAT4 has been identified as a susceptibility gene for RA. STAT4 is important in the signaling pathway through IL-12/IL-23 receptors, and is critical to the generation and maintenance of Th17 cells. STAT4 knockout mice are resistant to arthritis, and antisense STAT4 suppresses ongoing CIA.

There is increased expression of IL-22 and IL-22R in the synovium in RA, but the effects of this cytokine on cells of the joint are still unknown. Another cytokine that may play a role in Th17 biology is IL-15. IL-15 is overexpressed in RA synovial fluid and peripheral blood T cells, and can induce the secretion of IL-17 from PBMCs. RA synovial fibroblasts produce IL-15, which up-regulates IL-17 and TNF-α production by T cells. IL-17 and TNF-α then, in turn, stimulate production of IL-15 and IL-6 by the synovial fibroblasts (FLS), creating a positive feedback loop. In mice, insertion of an IL-15 transgene on an arthritis resistant strain increases severity of arthritis associated with increased IL-17 and IL-23 receptor expression, while an IL-15 receptor antagonist ameliorates CIA and down-regulates TNF-α, IL-1β, IL-6, and IL-17.

IL-17 can synergize with resting T cells to stimulate RA FLS to secrete IL-6, IL-8, and prostaglandin E ₂ . Cytokine-activated T cells can adhere avidly to RA FLS and induce IL-6 and IL-8 production from the FLS, which is further enhanced by exogenous IL-17. Neutralization of TNF-α, a cytokine that is both upstream and downstream of IL-17, inhibits the production of these proinflammatory cytokines, which are important in mediating joint inflammation in RA and CIA. T cells can also induce IL-6 production by osteoblasts, an effect that is augmented by IL-17. Such findings provide clues to pathways for in situ stimulation of T cells that could foster chronic inflammation in RA synovium.

Understanding the regulation of Th17 cells will be important in designing therapeutic options targeting IL-17. IL-4 is a potent suppressor of IL-17, both in vitro and in vivo. Dendritic cells genetically modified to express IL-4 have been shown to inhibit collagen-specific IL-17 production and reduce the incidence and severity of CIA. The suppression mediated by IL-4 dendritic cells is robust and not reversed by exogenous IL-23. Similarly, injection of an adenoviral vector expressing IL-4 has been shown to ameliorate CIA by down-regulating joint IL-17 levels and reducing bone and cartilage damage.

Overview of approaches to Th17 inhibition in RA

It is possible that current biologic and even nonbiologic disease-modifying antirheumatic drugs (DMARDs) used in RA work in part through effects (largely indirect) on Th17 cells, but much more evidence is needed to support this notion. For example, masking of CD28 ligands by CTLA-4Ig could deprive Th17 cells of a critical second activation signal early in their differentiation. Depletion of B cells by anti-CD20 could deprive Th17 cells of an important source of IL-6 while also diminishing the pools of antigen-presenting cells. Inhibition of IL-1 and possibly even TNF would tend to diminish Th17 differentiation. The subsequent paragraphs do not consider all of these approaches in the context of Th17 cells, but instead focus on the more recent strategies that neutralize IL-23/IL-12, block the IL-6 receptor, or inhibit IL-17 directly.

Anti–IL-12/IL-23

Ustekinumab is a human monoclonal antibody targeting the shared subunit p40 of IL-12 and IL-23. This antibody is being extensively tested in psoriasis, and clinical studies are also being undertaken in other diseases that may involve IL-12 and IL-23, such as psoriatic arthritis, Crohn disease, and multiple sclerosis. Inhibition of IL-23 is of great interest in diseases that involve pathogenic Th17 cells, while inhibition of IL-12 is logical in diseases that are mediated by Th1 cells, or both Th1 and Th17 cells, but could be counterproductive in a disease in which Th1 cells play a regulatory role vis-à-vis Th17 cells.

Safety and initial clinical efficacy was shown in phase 1 and 2 trials with ustekinumab in psoriasis. Two phase 3 studies have been done with ustekinumab in psoriasis: PHOENIX 1 and PHOENIX 2. Both PHOENIX 1 and PHOENIX 2 used similar doses of ustekinumab and similar trial design for the initial part of the study, but there were differences in the study design for the latter part of each study. In the initial phase of both studies patients received ustekinumab at 45 mg or 90 mg every 12 weeks, or placebo. Patients who were started on placebo were switched to the active drug at 12 weeks. In PHOENIX 1, patients were assessed for withdrawal of ustekinumab during the latter half of the study. In PHOENIX 2, effects of dose escalation of ustekinumab were assessed in partial responders from the initial part of this study. A significantly greater number of patients on ustekinumab achieved 75% improvement in the psoriasis area and severity index scores (PASI 75) compared with patients receiving placebo. In addition, patients who were started on placebo and subsequently switched to ustekinumab achieved a degree of clinical improvement similar to patients started on the active drug from the beginning of the study. Moreover, PHOENIX 1 showed that withdrawal of ustekinumab was associated with relapses that were responsive to retreatment with ustekinumab. PHOENIX 2 showed that in nonresponders to ustekinumab reducing the interval of drug administration from every 12 weeks to 8 weeks was beneficial for those who were on the 90-mg dosage from the start of the study. Patients who were on 45 mg of ustekinumab every 12 weeks did not have any further improvement in their PASI 75 responses on reducing the interval of dosing to every 8 weeks.

In a recently reported trial, ustekinumab was compared with etanercept in patients with plaque psoriasis. In this 12-week study patients were randomized to receive either etanercept (50 mg twice weekly) or ustekinumab 45 mg or 90 mg at weeks 0 and 4. The patients in the etanercept group who did not have significant improvement at week 12, received ustekinumab (2 doses, 4 weeks apart). In addition, patients who had received at least 1 cycle of ustekinumab and were experiencing relapses also received repeat doses of ustekinumab. The patients receiving ustekinumab had better therapeutic responses than those receiving etanercept. PASI 75 scores were seen in 67.5% of patients on 45 mg of ustekinumab, and in 73.8% of patients on 90 mg of ustekinumab versus 56.8% of patients on etanercept. Among the patients who did not have clinical improvement on etanercept and were switched to ustekinumab, 48.9% had improvement in their PASI 75 scores.

Ustekinumab has also shown promise in a trial involving patients with psoriatic arthritis. In this study 146 patients were randomized to either a ustekinumab group or a placebo group. Patients in the ustekinumab group received 63 to 90 mg of the drug weekly for the first 4 weeks followed by placebo on weeks 12 and 16. Patients assigned to the placebo group received placebo weekly for the first 4 weeks and then 63 to 90 mg of ustekinumab on weeks 12 and 16. All patients were followed for 36 weeks. During the first 12 weeks of the study, 52% of patients in the ustekinumab group achieved a PASI 75 response in comparison to 5% in the placebo group. Forty-two percent of patients in the ustekinumab group had achieved ACR20 (American College of Rheumatology 20% improvement measure) responses versus 14% in the placebo group in the first 12 weeks. At 12 weeks, the ACR50 and ACR70 responses were significantly better in the ustekinumab group than in the placebo group. ACR50 responses were 25% versus 7% in the ustekinumab and placebo groups, respectively. Similarly, the ACR70 responses were 11% and 0% in the ustekinumab and placebo groups, respectively. Patients who received placebo in the first 4 weeks then received 2 doses of ustekinumab at weeks 16 and 20 showed improvement in their ACR20 responses. The ACR20 responses in this group at 24 weeks was 51%, at 28 weeks 45%, and at 36 weeks 42%, which were very similar to those who had received the active drug from the start of the study. As stated previously, patients in the ustekinumab group received active drug for the first 4 weeks but were followed for 36 weeks. In these patients ACR20, ACR50, and ACR70 responses peaked at 16 to 20 weeks, and mostly stayed steady or had a very slow decline up to 36 weeks. The adverse events, including headaches, diarrhea, back pain, and nausea, were similar in the 2 groups. The rates of infections were similar overall, 36% in the ustekinumab group and 30% in the placebo group, and upper respiratory tract infection was 13% in the ustekinumab group versus 9% in the placebo group. At present there is a phase 3 study underway in patients with psoriatic arthritis (NCT01009086).

In a clinical trial involving patients with Crohn disease, administration of ustekinumab was associated with significantly better therapeutic improvement than placebo. Of note, in another clinical trial involving patients with relapsing-remitting multiple sclerosis, administration of ustekinumab was not associated with clinical improvement.

The immunologic mechanism underlying a therapeutic response to ustekinumab remains to be evaluated. Ustekinumab inhibited the production of IFN-γ and IL-17A by normal human peripheral blood mononuclear cells stimulated by IL-12 and IL-23. Further, there was inhibition of the up-regulation of cutaneous lymphocyte antigen, IL-2 receptor, IL-12 receptor, and CD40 ligand, and reduced secretion of TNF-α, IL-2, and IL-10. In the phase 1 study in psoriasis, a single dose of anti-p40 antibody led to down-regulation of both p40 and IL-23 p19 expression in skin lesions.

Data regarding efficacy of ustekinumab in RA are not yet available. It is possible that the approach of inhibiting both IL-12 and IL-23 action will be inferior in RA, compared with inhibition of IL-23 alone, if RA turns out to be primarily a Th17 disease rather than a mixed Th1/Th17 disease. It is also possible that the primary pathogenic Th subset in RA may differ between patients, or over time during the evolution of RA in an individual patient.

Anti–IL-6 receptor

The role of IL-6 in mediating inflammation in arthritis has been confirmed in several preclinical studies. Administration of anti–IL-6 receptor antibody (tocilizumab) reduced severity of inflammatory arthritis in mice as well as in monkeys. Mice with a point mutation in the regulatory pathway that controls signaling through the IL-6 receptor develop spontaneous arthritis due to prolonged unrestrained stimulation of this receptor. IL-6 is elevated in the synovial fluid and sera of patients with RA, and its level correlates with disease activity. IL-6 has effects on various cell types including neutrophils, T cells, B cells, monocytes, and osteoclasts. The IL-6 receptor is also expressed on hepatocytes and IL-6 can directly induce C-reactive protein (CRP) production from hepatocytes, thus elevating serum levels of CRP during inflammatory arthritis. The key role of IL-6 in Th17 cell development potentially places it as an upstream target in immune/inflammatory cascade in RA, along with its obvious role as a downstream target that is prominent in the effector phases of joint inflammation and damage.

A phase 1 study of tocilizumab in RA was not associated with significant adverse events. In a phase 2 study, 164 patients with refractory RA were randomized to receive MRA (the anti–IL-6R antibody now named tocilizumab), at 8 mg/kg, 4 mg/kg, or placebo every 4 weeks for 24 weeks. At 24 weeks patients receiving MRA had significantly improved clinical responses. The ACR20/50/70 responses were 78%/40%/16% in the MRA 8 mg/kg group, 57%/26%/20% in the MRA 4 mg/kg group, and 11%/1.9%/0% in the placebo group. DAS28 (disease activity score for 28 joints) scores also reflected similar improvements in the patients receiving MRA. Patients in the MRA (tocilizumab) arm of this trial were followed in an open-label extension for 5 years in the STREAM study. At the end of 5 years the ACR20/50/70 responses were 84%/69%/43.6%. The clinical efficacy peaked in the first year and remained at that level for the entire 5 years.

In contrast to the above trial, which compared tocilizumab with placebo, the CHARISMA trial was performed to compare the clinical efficacy of tocilizumab with that of methotrexate. Three hundred and fifty-nine patients were randomized to receive tocilizumab (at 2 mg/kg, 4 mg/kg, or 8 mg/kg every 4 weeks) alone or in combination with methotrexate for 16 weeks. The control group received methotrexate only. There was significant clinical improvement with tocilizumab alone or in combination with methotrexate. Among the groups that received tocilizumab alone, those patients who received 4 mg/kg or 8 mg/kg of tocilizumab had higher ACR20/50/70 responses when compared with the 2 mg/kg tocilizumab group or methotrexate alone group. The ACR20 responses were 63%, 61%, 31%, and 41% in the tocilizumab 8 mg/kg, 4 mg/kg, 2 mg/kg, and the methotrexate group, respectively. The ACR50 responses were higher in the tocilizumab 8 mg/kg group than in the 4 mg/kg group, 41% versus 28%. Similarly, the ACR70 responses were higher in the 8 mg/kg group; 16% versus 6% in the tocilizumab 4 mg/kg group. When tocilizumab was administered along with methotrexate, the ACR20/50/70 responses were significantly improved in all groups including the group receiving 2 mg/kg of tocilizumab. The ACR20/50/70 responses were 74%/53%/37% in the tocilizumab 8 mg/kg + methotrexate group; 63%/37%/12% in the tocilizumab 4 mg/kg + methotrexate group; 64%/32%/14% in the tocilizumab 2 mg/kg + methotrexate group in comparison with methotrexate alone, which was 41%/29%/16%. Similar improvements were seen when DAS28 scores were used to measure responses. The clinical improvement was apparent by 1 month after starting infusion and continued to increase during the 16-week study.

The phase 3 OPTION trial was similar to the CHARISMA trial except that this study was continued for 24 weeks. In this study, 623 patients were randomized into 3 groups: (i) 205 patients receiving tocilizumab at 8 mg/kg every 4 weeks + methotrexate, (ii) 214 patients receiving tocilizumab at 4 mg/kg every 4 weeks + methotrexate, and (iii) 204 patients on placebo + methotrexate. Patients were followed for 24 weeks. At the conclusion of this period patients on tocilizumab had significantly better responses than those on methotrexate alone: 59% of patients in the 8 mg/kg group, 48% of patients in the 4 mg/kg group, and 26% in the placebo group achieved ACR20 responses at 24 weeks. A similar trend was seen with the ACR50 and ACR70 responses, with 44% in the 8 mg/kg, 31% in the 4 mg/kg, and 11% in the placebo group achieving ACR50 responses. Twenty-two percent in the 8 mg/kg, 12% in the 4 mg/kg, and 2% in the placebo groups achieved ACR70 responses. In addition, 27% of patients in the 8 mg/kg and 13% in the 4 mg/kg groups compared with 0.8% in the placebo group achieved DAS28 responses of less than 2.6, consistent with remission. Therapeutic efficacy was seen after the first dose and continued to improve up to 24 weeks. There were improvements in the HAQ-DI (Health Assessment Questionnaire Disability Index) and fatigue scores as well.

In the phase 3 TOWARD trial the therapeutic efficacy of tocilizumab in combination with other DMARDs was assessed. In this trial, 1220 patients on stable DMARD doses were randomized to receive either tocilizumab 8 mg/kg every 4 weeks or placebo for 24 weeks. The patients receiving tocilizumab in combination with DMARDs had superior clinical efficacy compared with the DMARDs + placebo group. ACR20 responses were 61% in the tocilizumab + DMARD group versus 25% in the placebo + DMARD group. The ACR50/70 responses were 38%/21% in the tocilizumab group and 9%/3% in the placebo group. Thirty percent of patients in the tocilizumab group achieved DAS28 scores less than 2.6 versus 3% in the DMARD only group. The clinical responses were seen across all DMARDs, including methotrexate, leflunomide, sulfasalazine, hydroxychloroquine, and azathioprine. The rate of clinical improvement was similar whether patients were on 1, 2, or 3 DMARDs.

Furthermore, tocilizumab + methotrexate was shown to be effective in patients who had failed anti-TNF agents in the RADIATE trial. In this 24-week trial 499 patients who were refractory to anti-TNF therapy were randomized to receive tocilizumab 8 mg/kg + methotrexate, or tocilizumab 4 mg/kg + methotrexate, or placebo + methotrexate. The ACR20 responses were 50%/30.4%/10% in the 8 mg/kg, 4 mg/kg, and placebo groups, respectively. The clinical efficacy of tocilizumab was independent of the specific anti-TNF agent that was used previously. Similarly, the ACR50 responses were 28.8%, 16.8%, and 3.8% in the tocilizumab 8 mg/kg, 4 mg/kg, and placebo groups. The ACR70 responses were 12.4%, 5%, and 1.3% in the tocilizumab 8 mg/kg, 4 mg/kg, and placebo groups. The DAS28 remission (<2.6) was achieved in 30%, 7.6%, and 1.6% among the 8 mg/kg, 4 mg/kg, and the placebo groups, respectively.

In the AMBITION study, tocilizumab monotherapy was compared with methotrexate monotherapy in patients who had not failed methotrexate or anti-TNF agents in the past. In this 24-week study, 673 patients were randomized to receive tocilizumab 8 mg/kg every 4 weeks, methotrexate starting at 7.5 mg weekly, increasing to 20 mg in 8 weeks, or placebo for 8 weeks followed by tocilizumab 8 mg/kg weekly. The ACR20 responses were 70% in the tocilizumab group versus 52.5% in the methotrexate group. 33.6% of patients in the tocilizumab group achieved DAS28 scores of less than 2.6 versus 12.1% in the methotrexate group.

Tocilizumab has also been shown to slow radiographic bone damage in the SAMURAI trial. In this study, 306 patients were randomized to receive 8 mg/kg of tocilizumab weekly or conventional DMARDs for 52 weeks. At 52 weeks the mean total van der Heijde scores were lower in the tocilizumab group than in the DMARD group. In this study 306 patients with RA of less than 5 years’ duration were randomized to receive tocilizumab 8 mg/kg every 4 weeks or DMARDs for 52 weeks. The modified total Sharp score (TSS) at entry was 29.4. At the conclusion of the study, patients in the tocilizumab group had lower increments in their TSS than those in the DMARDs group (2.3 vs 6.1). Clinical improvement was similar to previous trials.

Finally, efficacy of tocilizumab was shown in methotrexate nonresponders in the SATORI trial. In this trial 127 patients were randomized to receive tocilizumab 8 mg/kg or weekly methotrexate (8 mg/wk—the most commonly used dosage in Japan) for 24 weeks. 80.3% of patients in the tocilizumab group achieved ACR20 responses compared with 25% in the control group. The ACR50 and ACR70 responses were 49.2% and 29.5%, respectively in the tocilizumab group compared with 10.9% and 6.3% in the control group. These differences in ACR responses were corroborated by improvements in the DAS28 responses. The clinical effects were evident at 4 weeks and continued up to 24 weeks.

In this trial, serum VEGF levels dropped by 346.9 pg/mL in the tocilizumab group compared with the control group, in which VEGF levels dropped by 74.0 pg/mL. VEGF is an angiogenic factor that can induce growth of new blood vessels required to sustain synovial hypertrophy and inflammation in RA. VEGF is produced by macrophages, vascular smooth muscle cells, synovial lining cells, neutrophils from synovial fluid, and peripheral blood mononuclear cells. VEGF can also facilitate migration of cells from blood into the synovium and thus maintain synovial inflammation. VEGF is increased in the sera of patients with RA.

A 2-year trial is currently underway to determine the safety and efficacy of tocilizumab + methotrexate in methotrexate nonresponders, the LITHE trial. In this trial, 1200 patients were randomized to receive tocilizumab 4 mg/kg + methotrexate, tocilizumab 8 mg/kg + methotrexate, or methotrexate + placebo. The dose of methotrexate is 10 to 25 mg weekly, which is higher than the previous SATORI trial where it was used at 8 mg weekly. Patients in the placebo arm were given the option of switching to blinded rescue therapy from week 16. At the initial 52-week analysis of this study, the ACR20 responses were 56%, 47%, and 25% in the tocilizumab 8 mg/kg, tocilizumab 4 mg/kg, and placebo groups, respectively. Similarly, the ACR50 and ACR70 responses were higher in the tocilizumab group, with the 8 mg/kg group showing higher responses than the 4 mg/kg group. The mean change in the Genant modified TSS from baseline was 0.29, 0.34, and 1.13 in the tocilizumab 8 mg/kg, tocilizumab 4 mg/kg, and placebo groups, respectively.