IL-6 in Inflammation, Immunity, and Disease
`
`Toshio Tanaka1,2, Masashi Narazaki3, and Tadamitsu Kishimoto4
`
`1Department of Clinical Application of Biologics, Osaka University Graduate School of Medicine, Osaka
`University, Osaka 565-0871, Japan
`2Department of Immunopathology, World Premier International Immunology Frontier Research Center, Osaka
`University, Osaka 565-0871, Japan
`3Department of Respiratory Medicine, Allergy and Rheumatic Diseases, Osaka University Graduate School
`of Medicine, Osaka University, Osaka 565-0871, Japan
`4Laboratory of Immune Regulation, World Premier International Immunology Frontier Research Center, Osaka
`University, Osaka 565-0871, Japan
`
`Correspondence: kishimoto@ifrec.osaka-u.ac.jp
`
`Interleukin 6 (IL-6), promptly and transiently produced in response to infections and tissue
`injuries, contributes to host defense through the stimulation of acute phase responses, he-
`matopoiesis, and immune reactions. Although its expression is strictly controlled by tran-
`scriptional and posttranscriptional mechanisms, dysregulated continual synthesis of IL-6
`plays a pathological effect on chronic inflammation and autoimmunity. For this reason,
`tocilizumab, a humanized anti-IL-6 receptor antibody was developed. Various clinical
`trials have since shown the exceptional efficacy of tocilizumab, which resulted in its approval
`for the treatment of rheumatoid arthritis and juvenile idiopathic arthritis. Moreover, tocili-
`zumab is expected to be effective for other intractable immune-mediated diseases. In this
`context, the mechanism for the continual synthesis of IL-6 needs to be elucidated to facilitate
`the development of more specific therapeutic approaches and analysis of the pathogenesis of
`specific diseases.
`
`IL-6 is a soluble mediator with a pleiotropic
`
`effect on inflammation, immune response,
`and hematopoiesis. At first, distinct functions
`of IL-6 were studied and given distinct names
`based on their biological activity. For example,
`the name B-cell stimulatory factor 2 (BSF-2)
`was based on the ability to induce differentia-
`tion of activated B cells into antibody (Ab)-pro-
`ducing cells (Kishimoto 1985), the name hepa-
`tocyte-stimulating factor (HSF) on the effect of
`acute phase protein synthesis on hepatocytes,
`
`the name hybridoma growth factor (HGF) on
`the enhancement of growth of fusion cells be-
`tween plasma cells and myeloma cells, or the
`name interferon (IFN)-b2 owing to its IFN an-
`tiviral activity. When the BSF-2 cDNA was suc-
`cessfully cloned in 1986 (Hirano et al. 1986),
`however, it was found that the molecules with
`different names studied by various groups were
`in fact identical, resulting in the single name IL-
`6 (Kishimoto 1989). Human IL-6 is made up of
`212 amino acids, including a 28-amino-acid
`
`Editor: Ruslan M. Medzhitov
`Additional Perspectives on Innate Immunity and Inflammation available at www.cshperspectives.org
`Copyright # 2014 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a016295
`Cite this article as Cold Spring Harb Perspect Biol 2014;6:a016295
`
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`T. Tanaka et al.
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`signal peptide, and its gene has been mapped to
`chromosome 7p21. Although the core protein
`is (cid:2)20 kDa, glycosylation accounts for the size
`of 21 – 26 kDa of natural IL-6.
`
`BIOLOGICAL EFFECT OF IL-6 ON
`INFLAMMATION AND IMMUNITY
`
`After IL-6 is synthesized in a local lesion in the
`initial stage of inflammation, it moves to the
`liver through the bloodstream,
`followed by
`the rapid induction of an extensive range of
`acute phase proteins such as C-reactive pro-
`tein (CRP), serum amyloid A (SAA), fibrinogen,
`haptoglobin, and a1-antichymotrypsin (Fig. 1)
`(Heinrich et al. 1990). On the other hand, IL-6
`reduces the production of fibronectin, albu-
`min, and transferrin. These biological effects
`on hepatocytes were at first studied as belong-
`ing to HSF. When high-level concentrations of
`SAA persist for a long time, it leads to a serious
`complication of several chronic inflammatory
`
`diseases through the generation of amyloid A
`amyloidosis (Gillmore et al. 2001). This results
`in amyloid fibril deposition, which causes pro-
`gressive deterioration in various organs. IL-6 is
`also involved in the regulation of serum iron and
`zinc levels via control of their transporters. As
`for serum iron, IL-6 induces hepcidin produc-
`tion, which blocks the action of iron transporter
`ferroportin 1 on gut and, thus, reduces serum
`iron levels (Nemeth et al. 2004). This means
`that the IL-6-hepcidin axis is responsible for hy-
`poferremia and anemia associated with chronic
`inflammation. IL-6 also enhances zinc importer
`ZIP14 expression on hepatocytes and so induces
`hypozincemia seen in inflammation (Liuzzi et
`al. 2005). When IL-6 reaches the bone marrow,
`it promotes megakaryocyte maturation, thus
`leading to the release of platelets (Ishibashi et
`al. 1989). These changes in acute phase protein
`levels and red blood cell and platelet counts are
`used for the evaluation of inflammatory sever-
`ity in routine clinical laboratory examinations.
`
`IL-6
`
`Hepatocyte
`
`CRP
`Serum amyloid A
`Fibrinogen
`Albumin
`Hepcidin
`
`Bone marrow
`
`Platelet
`
`CD4 T cell
`
`Th17 differentiation
`
`inhibition
`
`CD4 T cell
`
`Treg differentiation
`
`CD8 T cell
`
`B cell
`
`Synovial
`fibroblast
`
`Dermal
`fibroblast
`
`Cytotoxic T-cell
`differentiation
`
`Antibody
`production
`RANKL
`Osteoclast
`differentiation
`VEGF
`Angiogenesis
`Vascular permeability
`
`Collagen
`
`Amyloid A amyloidosis
`Cardiovascular event
`Edema
`Anemia of inflammation
`Thrombocytosis
`
`Autoimmunity
`chronic inflammation
`
`Autoantibody
`hypergammaglobulinemia
`
`Osteoporosis
`bone fracture
`
`Joint swelling
`edema
`
`Fibrosis
`
`Figure 1. IL-6 in inflammation, immunity, and disease. IL-6 is a cytokine featuring pleiotropic activity; it
`induces synthesis of acute phase proteins such as CRP, serum amyloid A, fibrinogen, and hepcidin in hepato-
`cytes, whereas it inhibits production of albumin. IL-6 also plays an important role on acquired immune response
`by stimulation of antibody production and of effector T-cell development. Moreover, IL-6 can promote differ-
`entiation or proliferation of several nonimmune cells. Because of the pleiotropic activity, dysregulated continual
`production of IL-6 leads to the onset or development of various diseases. Treg, regulatory T cell; RANKL,
`receptor activator of nuclear factor kB (NF-kB) ligand; VEGF, vascular endothelial growth factor.
`
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`Furthermore, IL-6 promotes specific differ-
`þ
`entiation of naı¨ve CD4
`T cells, thus performing
`an important function in the linking of innate
`to acquired immune response. It has been shown
`that IL-6, in combination with transforming
`growth factor (TGF)-b, is indispensable for
`þ
`Th17 differentiation from naı¨ve CD4
`T cells
`(Korn et al. 2009), but that IL-6 also inhibits
`TGF-b-induced Treg differentiation (Bettelli
`et al. 2006). Up-regulation of the Th17/Treg
`balance is considered to be responsible for the
`disruption of immunological tolerance, and is
`thus pathologically involved in the development
`of autoimmune and chronic inflammatory dis-
`eases (Kimura and Kishimoto 2010). It has been
`further shown that IL-6 also promotes T-follic-
`ular helper-cell differentiation as well as produc-
`tion of IL-21 (Ma et al. 2012), which regulates
`immunoglobulin (Ig) synthesis and IgG4 pro-
`duction in particular. IL-6 also induces the dif-
`þ
`T cells into cytotoxic T
`ferentiation of CD8
`cells (Okada et al. 1988). Under one of its previ-
`ous names, BSF-2, IL-6 was found to be able to
`induce the differentiation of activated B cells
`into Ab-producing plasma cells, so that contin-
`uous oversynthesis of IL-6 results in hypergam-
`maglobulinemia and autoantibody production.
`IL-6 exerts various effects other than those
`on hepatocytes and lymphocytes and these are
`frequently detected in chronic inflammatory
`diseases (Kishimoto 1989; Hirano et al. 1990;
`Akira et al. 1993). One of these effects is that,
`when IL-6 is generated in bone marrow stromal
`cells, it stimulates the RANKL (Hashizume et al.
`2008), which is indispensable for the differenti-
`ation and activation of osteoclasts (Kotake et
`al. 1996), and this leads to bone resorption and
`osteoporosis (Poli et al. 1994). IL-6 also induces
`excess production of VEGF, leading to enhanced
`angiogenesis and increased vascular permeabil-
`ity, which are pathological features of inflam-
`matory lesions and are seen in, for example,
`synovial tissues of rheumatoid arthritis (RA)
`or edema of remitting seronegative symmetri-
`cal synovitis with pitting edema (RS3PE) syn-
`drome (Nakahara et al. 2003; Hashizume et al.
`2009). Finally, it has been reported that IL-6
`aids keratinocyte proliferation (Grossman et al.
`1989) or the generation of collagen in dermal
`
`IL-6 in Inflammation, Immunity, and Disease
`
`fibroblasts that may account for changes in the
`skin of patients with systemic sclerosis (Duncan
`and Berman 1991).
`
`REGULATION OF IL-6 SYNTHESIS
`
`IL-6 functions as a mediator for notification of
`the occurrence of some emergent event. IL-6 is
`generated in an infectious lesion and sends out a
`warning signal to the entire body. The signature
`of exogenous pathogens, known as pathogen-
`associated molecular patterns, is recognized in
`the infected lesion by pathogen-recognition re-
`ceptors (PRRs) of immune cells such as mono-
`cytes and macrophages (Kumaret al. 2011). These
`PRRs comprise Toll-like receptors (TLRs), ret-
`inoic acid-inducible gene-1-like receptors, nu-
`cleotide-binding oligomerization domain-like
`receptors, and DNA receptors. They stimulate
`a range of signaling pathways including NF-kB,
`and enhance the transcription of the mRNA
`of inflammatory cytokines such as IL-6, tumor
`necrosis factor (TNF)-a, and IL-1b. TNF-a and
`IL-1b also activate transcription factors to pro-
`duce IL-6.
`IL-6 also issues a warning signal in the event
`of tissue damage. Damage-associated molecular
`patterns (DAMPs), which are released from
`damaged or dying cells in noninfectious inflam-
`mations such as burn or trauma, directly or
`indirectly promote inflammation. During ster-
`ile surgical operations, an increase in serum IL-
`6 levels precedes elevation of body temperature
`and serum acute phase protein concentration
`(Nishimoto et al. 1989). DAMPs from injured
`cells contain a variety of molecules such as mi-
`tochondrial (mt) DNA, high mobility group
`box 1 (HMGB1), and S100 proteins (Bianchi
`2007). Serum mtDNA levels in trauma patients
`are thousands of times higher than in con-
`trols and this elevation leads to TLR9 stimula-
`tion and NF-kB activation (Zhang et al. 2010),
`whereas binding of HMGB1 to TLR2, TLR4,
`and the receptor of advanced glycation end
`products (RAGE) can promote inflammation.
`The S100 family of proteins comprises more
`than 25 members, some of which also interact
`with RAGE to evoke sterile inflammation (Sims
`et al. 2010).
`
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`T. Tanaka et al.
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`In addition to immune-mediated cells, mes-
`enchymal cells, endothelial cells, fibroblasts, and
`many other cells are involved in the production
`of IL-6 in response to various stimuli (Akira
`et al. 1993). The fact that IL-6 issues a warning
`signal to indicate occurrence of an emergency
`accounts for the strict regulation of IL-6 synthe-
`sis both gene transcriptionally and posttran-
`scriptionally. A number of transcription factors
`have been shown to regulate the IL-6 gene tran-
`scription (Fig. 2). The functional cis-regula-
`0
`flank-
`tory elements in the human IL-6 gene 5
`ing region are found binding sites for NF-kB,
`specificity protein 1 (SP1), nuclear factor IL-6
`(NF-IL-6) (also known as CAAT/enhancer-
`binding protein b), activator protein 1 (AP-1),
`
`and interferon regulatory factor 1 (Libermann
`and Baltimore 1990; Akira and Kishimoto 1992;
`Matsusaka et al. 1993). Activation of cis-regu-
`latory elements by stimulation with IL-1, TNF,
`TLR-mediated signal, and forskolin lead to acti-
`vation of the IL-6 promoter.
`A polymorphism at position -174 of the IL-6
`promoter region is reportedly associated with
`systemic onset
`juvenile idiopathic arthritis
`(Fishman et al. 1998) and susceptibility to RA
`in Europeans (Lee et al. 2012). Stimulation with
`lipopolysaccharide (LPS) and IL-1 did not evoke
`any response in a reporter assay using -174 C
`construct. A -174 G construct, on the other
`hand, was found to promote transcription of
`the reporter gene, suggesting that a genetic back-
`
`MicroRNA
`
`Promotion
`
`Protein
`NF-κB, SP-1
`NF-IL-6, AP-1
`IRF1, Tax
`TAT, HBVX
`Mutant p53
`Repression Ahr,GR, ER miR-155 (targeting NF-IL-6)
`p53, Rb
`miR-146a/b (targeting IRAK1)
`PPARα
`miR-223 (targeting STAT3)
`
`Transcriptional regulation
`
`Stabilization
`
`Degradation
`
`MicroRNA
`
`miR-365
`miR-608
`
`Protein
`Arid5a
`P38α
`ORF57
`Regnase-1
`TTP
`BRF1
`BRF2
`
`Posttranscriptional regulation
`
`AP-1
`IRF1
`-283 ∼ -227 -267 ∼ -254
`
`E
`M R
`1 ∼ - 1
`5
`- 1
`
`3
`
`7
`
`F -I L - 6
`5 ∼ - 1
`
`4
`
`8
`
`5
`
`N
`- 1
`
`SP-1
`-123 ∼ -119
`
`SP-1
`-108 ∼ -104
`
`5′-AUUUUAAUUAUUUUUAAUUUAUUGAUAAUUUAAAUAAGU
`AAA CUUUAAGUUAAUUUAUGAUUGAUAUUUAUUAUUUUUA-3′
`AU-rich elements
`
`5′
`
`3′
`
`5′UTR
`
`IL-6 mRNA
`
`3′UTR
`
`NF-IL-6 NF-κB
`-87 ∼ -76
`-75 ∼ -63
`
`Figure 2. Transcriptional and posttranscriptional regulation of IL-6 gene. The expression and degradation of IL-
`6 mRNA is regulated transcriptionally and posttranscriptionally by several proteins and microRNAs. Activation
`of these proteins and microRNAs determines the fate of IL-6 mRNA. NF-IL-6, nuclear factor of IL-6; Tax,
`transactivator protein; TAT, transactivator of the transcription; HBVX, hepatitis B virus X protein; Ahr, aryl
`hydrocarbon receptor; GR, glucocorticoid receptor; ER, estrogen receptor; Rb, retinoblastoma; PPARa, perox-
`isome proliferator– activated receptor a; miR, microRNA; IRAK1, IL-1 receptor– associated kinase 1; STAT3,
`signal transducer and activator of transcription 3; ORF, open reading frame; TTP, tristetraprolin; BRF1, butyrate
`response factor 1.
`
`4
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`ground of excess IL-6 production constitutes
`a risk factor for juvenile idiopathic arthritis
`and RA.
`An interesting finding is that some viral
`products enhance the DNA-binding activity of
`NF-kB and NF-IL-6, resulting in an increase in
`IL-6 mRNA transcription. An instance of this
`phenomenon is that interaction with NF-kB of
`the Tax derived from the human T lymphotropic
`virus 1 enhances IL-6 production (Ballard et al.
`1988; Leung and Nabel 1988). Another example
`is the enhancement of both NF-kB and NF-IL-6
`DNA-binding activity by the transactivator of
`the TAT protein of the human immunodeficien-
`cy virus 1 (Scala et al. 1994; Ambrosino et al.
`1997). Moreover, it has been shown that DNA
`binding of NF-IL-6 can be enhanced by the hu-
`man hepatitis B virus X protein (Mahe et al.
`1991; Ohno et al. 1999).
`On the other hand, some transcription fac-
`tors suppress IL-6 expression. Peroxisome pro-
`liferator– activated receptors (PPARs) are li-
`gand-activated transcription factors consisting
`of three subtypes: a, b, and g. Among three
`PPARs, fibrates-activated PPARa interacts with
`c-Jun and p65 NF-kB subunits, which negatively
`regulate IL-6 transcription (Delerive et al. 1999).
`In addition, some hormone receptors have been
`identified as repressors of IL-6 expression. The
`increase in serum IL-6 after menopause or ovar-
`ectomy is reportedly associated with suppres-
`sion of IL-6 expression by estrogen receptors
`(Jilka et al. 1992), whereas activation of the glu-
`cocorticoid receptor can repress IL-6 expression,
`and this is thought to be one of mechanisms
`responsible for the anti-inflammatory effects
`of corticosteroids (Ray and Prefontaine 1994).
`It has further been shown that retinoblastoma
`protein and p53 repress the IL-6 gene promoter,
`whereas it is up-regulated by mutant p53 (San-
`thanam et al. 1991).
`In addition, some microRNAs directly or
`indirectly regulate transcription activity. Inter-
`0
`untrans-
`action of microRNA-155 with the 3
`lated regions (UTR) of NF-IL-6 results in sup-
`pression of NF-IL-6 expression (He et al. 2009),
`whereas microRNA-146a/b and -223 indirect-
`ly suppress transcription of IL-6 by respec-
`tively targeting IL-1 receptor– associated kinase
`
`IL-6 in Inflammation, Immunity, and Disease
`
`1 and STAT3 (Chen et al. 2012; Zilahi et al.
`2012).
`
`PRODUCTION AND FUNCTION OF IL-6
`AND ARYL HYDROCARBON RECEPTOR
`
`Aryl hydrocarbon receptor (Ahr) not only
`affects IL-6 transcription, but also regulates in-
`nate and acquired immune response. Ahr, also
`known as the dioxin receptor, is a ligand-acti-
`vated transcription factor that belongs to the
`basic helix-loop-helix PER-ARNT-SIM family
`(Burbach et al. 1992; Ema et al. 1992). Ahr
`is present in the cytoplasm, where it forms a
`complex with Ahr-interacting protein (Bell
`and Poland 2000). On binding with a ligand,
`Ahr moves to the nucleus and dimerizes with
`the Ahr nuclear translocator (Arnt). Within the
`nucleus, the Ahr/Arnt heterodimer then binds
`to the xenobiotic response element (XRE),
`which leads to various toxicological effects (Fu-
`jii-Kuriyama et al. 1994; Dragan and Schrenk
`2000; Ohtake et al. 2003; Puga et al. 2005). Al-
`though the physiological ligands for Ahr are
`not well known, indoleamine 2,3-dioxygenase
`(IDO), which catalyzes tryptophan into kynu-
`renine, is induced by Ahr signaling and kynu-
`renine is one of the ligands of Ahr (Vogel et al.
`2008; Jux et al. 2009).
`An animal model of RA was used to show
`the essential role of Ahr in the induction of
`Th17 cells and Th17-dependent collagen-in-
`duced arthritis (CIA) (Kimura et al. 2008; Na-
`kahama et al. 2011). Stimulation of naı¨ve T cells
`with IL-6 plus TGF-b (Th17 cell – inducing
`condition) induced Ahr expression and dele-
`tion of the Ahr gene nullified the induction of
`Th17 cells.
`Ahr interacted with and inhibited the ac-
`tivities of STAT1 or STAT5, which mediate the
`anti-inflammatory signals of IL-27 and IFN-
`g, or IL-2, respectively (Harrington et al. 2005;
`Stumhofer et al. 2006; Laurence et al. 2007;
`Kimura et al. 2008), thus suppressing the inhib-
`itory signals for the induction of Th17 cells.
`Retinoid-related orphan receptors (ROR) g
`and a, which are activated by STAT3, are essen-
`tial transcription factors for Th17-cell induction
`(Ivanov et al. 2006; Yang et al. 2008) and Ahr was
`
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`T. Tanaka et al.
`
`found not to affect the ROR-g and -a expres-
`sion. In the Ahr gene – deficient mice, no arthri-
`tis developed in CIA. Moreover, with T-cell-spe-
`cific deletion of the Ahr gene, no development of
`CIAwas observed (Nakahama et al. 2011). These
`results clearly show that CIA is a T-cell-depen-
`dent disease and the presence of Ahr is essential
`for its development. In these Ahr-deficient mice,
`the number of Th17 cells decreased and that of
`Th1 cells increased but no significant changes
`were observed in Foxp3-expressing Treg cells.
`Ahr also regulates Th17-cell
`induction
`through regulation of microRNAs. In our study,
`Ahr-induced microRNA-132/212 cluster under
`Th17 cell – inducing conditions and transfection
`of microRNA-212 into naı¨ve T cells under these
`circumstances augmented the expression of IL-
`17-related genes such as IL-17A, IL-22, and IL-
`23R (Nakahama et al. 2013). One of the target
`genes of this microRNA is B-cell lymphoma 6,
`which is known as an inhibitor of Th17-cell in-
`duction (Yu et al. 2009). All of these findings
`show that Ahr accelerates inflammation through
`the enhancement of Th17-cell induction by sev-
`eral mechanisms.
`Interestingly, Ahr showed a negative reg-
`ulatory effect on peritoneal macrophages and
`bone marrow – derived dendritic cells (BMDC)
`(Nguyen et al. 2010). In the absence of Ahr,
`LPS-induced production of inflammatory cy-
`tokines such as IL-6, TNF, and IL-12 showed
`major increases in macrophages,
`indicating
`that Ahr negatively regulates inflammatory cy-
`tokine production. Ahr interacts with STAT1
`and NF-kB and the resultant complex of Ahr/
`STAT1 and NF-kB leads to inhibition of the
`promoter activity of IL-6 and other inflamma-
`tory cytokines (Kimura et al. 2009). In BMDC,
`Ahr is required for the activation of IDO leading
`to kynurenine production because the deletion
`of Ahr in BMDC leads to loss of IL-10 and ky-
`nurenine production. Coculture of naı¨ve T cells
`with Ahr-deficient BMDC in the presence of
`LPS resulted in reduction of Treg-cell induction,
`whereas addition of kynurenine rescued the in-
`duction of Treg cells by BMDC (Nguyen et al.
`2010). These findings indicate that Ahr is re-
`quired for the regulatory BMDC cells through
`the induction of IDO.
`
`STABILIZATION AND DEGRADATION OF
`IL-6 MRNA (ARID5A AND REGNASE-1)
`
`As for posttranscriptional regulation of cyto-
`kine expression, cytokine mRNA is controlled
`0
`0
`through both the 5
`and 3
`UTR (Chen and Shyu
`1995; Anderson 2008). Initiation of mRNA
`0
`UTR, and
`translation is determined by the 5
`0
`the stability of mRNA by the 3
`UTR. IL-6
`mRNA is regulated by modulation of AU-rich
`0
`UTR region, whereas
`elements located in the 3
`a number of RNA-binding proteins and micro-
`0
`UTRs and regulate the
`RNAs bind to the 3
`stability of IL-6 mRNA (Fig. 2). For example,
`IL-6 mRNA stabilization is promoted by mito-
`gen-activated protein kinase (MAPK) p38a via
`0
`UTRs of IL-6 (Zhao et al. 2008), and the sta-
`3
`bilization of both viral and human IL-6 mRNA
`by the Kaposi’s sarcoma – associated herpesvi-
`rus (KSHV) ORF-57 by competing with the
`binding of microRNA-1293 to the viral or
`of microRNA-608 to the human IL-6 mRNA
`(Kang et al. 2011). RNA-binding proteins,
`such as TTP and BRF1 and 2, on the other
`hand, promote IL-6 mRNA degradation (Pala-
`nisamy et al. 2012), whereas IL-6 mRNA levels
`are reduced by microRNAs such as microRNA-
`365 and -608 through direct interaction with IL-
`0
`UTR (Kang et al. 2011; Xu et al. 2011).
`6 3
`It was recently found that a nuclease known
`as regulatory RNase-1 (regnase-1) (also known
`as Zc3h12a) plays a part in the destabilization
`of IL-6 mRNA, and that the relevant knockout
`mice spontaneously develop autoimmune dis-
`eases accompanied by splenomegaly and lymph-
`adenopathy (Matsushita et al. 2009). The in-
`hibitor of NF-kB (IkB) kinase (IKK) complex
`controls IL-6 mRNA stability by phosphorylat-
`ing regnase-1 in response to IL-1R/TLR stim-
`ulation (Iwasaki et al. 2011). Phosphorylated
`regnase-1 underwent ubiquitination and degra-
`dation. Regnase-1 re-expressed in IL-1R/TLR-
`activated cells was found to feature delayed ki-
`netics, and regnase-1 mRNA to be negatively
`regulated by regnase-1 itself via a stem-loop re-
`0
`UTR. These find-
`gion present in the regnase-1 3
`ings show that IKK complex phosphorylates not
`only IkBa, activating transcription, but also reg-
`nase-1, releasing the brake on IL-6 mRNA ex-
`
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`pression. Regnase-1 also regulates the mRNAs of
`a set of genes, including c-Rel, Ox40, and IL-2
`0
`through cleavage of their 3
`UTRs in T cells. T-
`cell receptor engagement then leads to cleavage
`of regnase-1, which frees T cells from regnase-
`mediated suppression, thus indicating that reg-
`nase-1 may play a crucial role in T-cell activation
`(Uehata et al. 2013).
`We have recently identified a novel RNA-
`binding protein, AT-rich interactive domain-
`containing protein 5a (Arid5a), which binds
`0
`UTR of IL-6 mRNA, resulting in the
`to the 3
`selective stabilization of IL-6 but not of TNF-a
`or IL-12 mRNA (Masuda et al. 2013). Arid5a
`expression was found to be enhanced in macro-
`phages in response to LPS, IL-1b, and IL-6, and
`also to be induced under Th17-polarizing con-
`ditions in T cells. We also found that Arid5a gene
`deficiency inhibited elevation of IL-6 levels in
`LPS-injected mice and preferential Th17-cell
`development in experimental autoimmune en-
`cephalomyelitis. Moreover, Arid5a counteracted
`the destabilizing function of regnase-1 on IL-6
`mRNA (Fig. 3), indicating that the balance be-
`tween Arid5a and regnase-1 plays an important
`
`IL-6 in Inflammation, Immunity, and Disease
`
`role in IL-6 mRNA stability. All of these results
`suggest that posttranscriptional regulation of
`IL-6 mRNA by Arid5a and regnase-1 may play
`an important role in the expression of IL-6 and
`that the predominance of Arid5a over regnase-1
`promotes inflammatory processes and possibly
`induces the development of autoimmune in-
`flammatory diseases.
`During the so-called “cytokine storm,” a po-
`tentially fatal immune reaction induced by hy-
`peractivation of T cells, a major boost in IL-6
`production is observed but without comparable
`production of other inflammatory cytokines. A
`recent study showed that the cytokine storm
`induced by cancer immunotherapy using T-
`cell transfection was counteracted by the anti-
`IL-6 receptor antibody, tocilizumab (Grupp et
`al. 2013). Experimentally, inhalation by mice of
`peroxidized phospholipids induced a cytokine
`storm resulting from a greatly marked increase
`in the production of IL-6 but not TNF (Imai
`et al. 2008).
`These results showing the IL-6-specific ele-
`vation without any effect on the other inflam-
`matory cytokines strongly suggest the impor-
`
`LPS
`
`MD2
`
`TLR4
`
`IL-6
`
`MyD88
`
`IκΒ
`
`NF-κΒ
`
`IL-6
`Arid5a
`Regnase-1
`
`NF-κΒ
`
`mRNA
`
`Arid5a
`
`Regnase-1
`
`5′UTR
`
`IL-6 mRNA
`
`3′UTR
`
`Regnase-1
`
`5′UTR
`
`IL-6 mRNA
`degradation
`
`3′UTR
`
`Figure 3. IL-6 synthesis and regulation of IL-6 mRNA stability by Arid5a. Pathogen-associated molecular
`patterns are recognized by pathogen-recognition receptors to induce proinflammatory cytokines; in this figure,
`TLR4 recognizes LPS and induces IL-6 mRNA via activation of the NF-kB signaling pathway. Regnase-1
`promotes IL-6 mRNA degradation, whereas Arid5a inhibits destabilizing effects of regnase-1. The balance
`between Arid5a and regnase-1 is important for the regulation of IL-6 mRNA. MD2, myeloid differentiation
`protein 2; MyD88, myeloid differentiation primary response 88; IkB, inhibitor of NF-kB.
`
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`
`7
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`T. Tanaka et al.
`
`tance of posttranscriptional modification of IL-
`6 mRNA by Arid5a and regnase-1. The balance
`between Arid5a and regnase-1 may determine
`the pathological increase of IL-6 in various dis-
`eases including autoimmunity and even cyto-
`kine storm.
`
`IL-6 RECEPTOR–MEDIATED SIGNALING
`SYSTEM
`
`The IL-6 receptor– signaling system is made up
`of two receptor chains and downstream sig-
`naling molecules (Kishimoto et al. 1992). The
`IL-6 receptor (IL-6R) constitutes the IL-6-bind-
`ing chain, which occurs in two forms, 80 kDa
`transmembrane and 50 – 55 kDa – soluble IL-6R
`(sIL-6R), whereas 130 kDa gp130 constitutes
`the signal-transducing chain. Both of these
`proteins belong to the cytokine receptor family
`with a Trp-Ser-X-Trp-Ser motif (Yamasaki et al.
`1988; Hibi et al. 1990). sIL-6R without the cy-
`toplasmic region is present in human serum and
`after IL-6 binding to sIL-6R; the resultant com-
`plex induces the IL-6 signal on gp130-express-
`ing cells (Narazaki et al. 1993). The pleiotropic
`effect of IL-6 on various cells derives from the
`broad range of gp130 expression observed on
`cells (Taga and Kishimoto 1997). After binding
`
`of IL-6 to IL-6R, the IL-6/IL-6R complex in turn
`induces homodimerization of gp130 and trig-
`gers a downstream signal cascade (Fig. 4) (Mu-
`rakami et al. 1993). The activated IL-6 receptor
`complex is generated in the form of a hexamer-
`ic structure comprising two molecules each of
`IL-6, IL-6R, and gp130 (Boulanger et al. 2003).
`Of these components, IL-6R is a unique bind-
`ing-receptor for IL-6, whereas the signal-trans-
`ducing chain gp130 is shared by members of the
`IL-6 family of cytokines, that is, leukemia in-
`hibitory factor, oncostatin M, ciliary neurotro-
`phic factor, IL-11, cardiotrophin 1, cardiotro-
`phin-like cytokine, IL-27, and IL-35. Although
`all of these cytokines thus bind to their specific
`binding receptors, they use the same gp130 for
`their signals (Kishimoto et al. 1994, 1995). The
`only exception is virus-encoded IL-6, which is
`the product of KSHV (also known as human
`herpesvirus 8), and directly binds to and acti-
`vates gp130 (Aoki et al. 2001). The mechanism
`that the IL-6 cytokine family members use to
`employ the common signal transducer makes
`it clear why members of the IL-6 family show
`functional redundancy. The molecular elucida-
`tion of the IL-6-signaling system finally solved
`the long-standing mystery of why cytokines fea-
`tured pleiotropy and redundancy.
`
`IL-6
`
`Tocilizumab
`
`IL-6 receptor
`
`Soluble IL-6
`receptor
`
`gp130
`
`JAKs
`
`STAT3
`
`SHP-2
`
`MAPKs
`
`Figure 4. IL-6 receptor system and IL-6 blocker, a humanized anti-IL-6 receptor antibody tocilizumab. IL-6
`binds to soluble and transmembrane IL-6R and the complex, then induces homodimerization of gp130, leading
`to activation of the signaling system. A humanized anti-IL-6R antibody, tocilizumab, blocks IL-6-mediated
`signaling pathway by its inhibition of IL-6 binding to both receptors. JAKs, Janus kinases; SHP-2, SH2-domain
`containing protein tyrosine phosphatase-2.
`
`8
`
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`
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`Activation of gp130 in turn triggers activa-
`tion of downstream signaling molecules, that is,
`the Janus kinase (JAK)-STAT3 pathway and the
`JAK-SHP-2-mitogen-activated protein (MAP)
`kinase pathway. The regulation of various sets
`of IL-6 responsive genes, including acute phase
`proteins, is accounted for by the transcription
`factor STAT3, which also induces the suppressor
`of cytokine signaling 1 (SOCS1) and SOCS3,
`which share the SH2-domain. In this context,
`SOCS1 binds to tyrosine-phosphorylated JAK
`(Naka et al. 1997), whereas SOCS3 binds to
`tyrosine-phosphorylated gp130 (Schmitz et al.
`2000) to stop IL-6 signaling by means of a neg-
`ative feedback loop.
`
`IL-6 AND DISEASE
`
`An immediate and transient expression of IL-6
`is generated in response to environmental stress
`factors such as infections and tissue injuries.
`This expression triggers an alarm signal and ac-
`tivates host defense mechanisms against stress.
`Removal of the source of stress from the host
`is followed by cessation of IL-6-mediated acti-
`vation of the signal-transduction cascade by
`negative regulatory systems such as ligand-in-
`duced internalization and degradation of gp130
`and recruitment of SOCS (Naka et al. 1997), as
`well as degradation of IL-6 mRNA by regnase-1
`leading to termination of IL-6 production.
`However, dysregulated and persistent IL-6 pro-
`duction of mostly unknown etiology, one of
`which may be the unbalance between Arid5a
`and regnase-1, in certain cell populations leads
`to the development of various diseases. This
`association of IL-6 with disease development
`was first shown in a case of cardiac myxoma.
`The culture of fluid obtained from the myxoma
`tissues of a patient who presented with fever,
`polyarthritis with positivity for antinuclear fac-
`tor, elevated CRP level, and hypergammaglo-
`bulinemia, contained a large quantity of IL-6,
`which suggested that IL-6 may contribute to
`chronic inflammation and autoimmunity (Hi-
`rano et al. 1987). Subsequent studies have shown
`that dysregulation of IL-6 production occurs in
`the synovial cells of RA (Hirano et al. 1988),
`swollen lymph nodes of Castleman’s disease
`
`IL-6 in Inflammation, Immunity, and Disease
`
`(Yoshizaki et al. 1989), myeloma cells (Kawano
`et al. 1988), and peripheral blood cells or in-
`volved tissues in various other autoimmune
`and chronic inflammatory diseases and even
`malignant cells in cancers (Nishimoto et al.
`1989, 2005).
`Moreover, the pathological role of IL-6 in
`disease development has been shown in numer-
`ous animal models of diseases as well as the fact
`that IL-6 blockade by means of gene knockout or
`administration of anti-IL-6 or anti-IL-6R Ab
`can result in the preventive or therapeutic sup-
`pression of disease development. For example,
`IL-6 blockade resulted in a noticeable reduc-
`tion in susceptibility to Castleman’s disease –
`like symptoms in IL-6 transgenic mice (Katsume
`et al. 2002). Similar effects were observed in
`models of RA (Alonzi et al. 1998; Ohshima et
`al. 1998; Fujimoto et al. 2008), systemic lupus
`erythematosus (Mihara et al. 1998), systemic
`sclerosis (Kitaba et al. 2012), inflammatory my-
`opathies (Okiyama et al. 2009), experimental
`autoimmune uveoretinitis (Haruta et al. 2011),
`experimental autoimmune encephalomyelitis
`(Serada et al. 2008), and many other diseases.
`
`IL-6 TARGETING AS STRATEGY FOR
`TREATMENT OF IMMUNE-MEDIATED
`DISEASES
`
`In view of the range of biological activities of IL-
`6 and its pathological role in various diseases
`described above, it was anticipated that IL-6 tar-
`geting would constitute a novel treatment strat-
`egy for various immune-mediated diseases. The
`development of tocilizumab was a direct r