IL-6 in autoimmune disease and chronic inflammatory proliferative disease

Cytokine & Growth Factor Reviews 13 (2002) 357–368

Survey

IL-6 in autoimmune disease and chronic inflammatory proliferative disease Katsuhiko Ishihara a,b , Toshio Hirano a,b,c,∗ a

Department of Molecular Oncology (C7), Graduate School of Medicine, Osaka University, 2-2 Yamada-oka Suita, Osaka 565-0871, Japan b Laboratory of Developmental Immunology, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan c Laboratory for Cytokine Signaling, RIKEN Research Center for Allergy and Immunology (RCAI), Yokohama, Kanagawa, Japan

Abstract Interleukin 6 (IL-6), which was originally identified as a B-cell differentiation factor, is now known to be a multifunctional cytokine that regulates the immune response, hematopoiesis, the acute phase response, and inflammation. Deregulation of IL-6 production is implicated in the pathology of several disease processes. The expression of constitutively high levels of IL-6 in transgenic mice results in fatal plasmacytosis, which has been implicated in human multiple myeloma. Increased IL-6 levels are also observed in several diseases, including rheumatoid arthritis (RA), systemic-onset juvenile chronic arthritis (JCA), osteoporosis, and psoriasis. IL-6 is critically involved in experimentally induced autoimmune disease, such as antigen-induced arthritis (AIA), and experimental allergic encephalomyelitis. All these clinical data and animal models suggest that IL-6 plays critical roles in the pathogenesis of autoimmune diseases. Here we review the evidence for the involvement of IL-6 in the pathophysiology of autoimmune diseases and chronic inflammatory proliferative diseases (CIPD) and discuss the possible molecular mechanisms of its involvement. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Interleukin 6; Signal transduction; Autoimmune disease; Chronic inflammatory proliferative disease (CIPD)

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. IL-6 and chronic inflammation: its implication in polyclonal B-cell activation and autoantibody production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Overexpression of IL-6 results in the generation of polyclonal plasmacytosis and malignant plasmacytoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Insulin-dependent diabetes mellitus and IL-6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Inflammatory bowel disease and IL-6. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Experimental autoimmune encephalomyelitis and IL-6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Rheumatoid arthritis and IL-6. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Polymorphisms in the human IL-6 gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Possible mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. Conclusion and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Interleukin 6 (IL-6) was originally identified as a B-cell differentiation factor [1,2], but it is now known to be a multifunctional cytokine that regulates the immune response, ∗ Corresponding author. Tel.: +81-6-6879-3880; fax: +81-6-6879-3889. E-mail address: [email protected] (T. Hirano).

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hematopoiesis, the acute phase response, and inflammation [3]. IL-6 is a cytokine with a helical structure that is similar to many other cytokines [1,4]. The IL-6 receptor (R) and many other cytokine receptors are structurally similar and constitute the cytokine receptor super family [5–7]. In addition, cytokine receptor subunits are shared among several cytokine receptors [8–13]. This sharing of subunits is one of the mechanisms through which the functional redundancy of

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cytokine activities occurs. The IL-6 receptor system consists of two molecules, IL-6R␣ and gp130, the latter of which is shared among the receptors for IL-6, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), oncostatin M (OSM), IL-11, and cardiotrophin-1 (CT-1) [14]. IL-6 stimulation activates JAK tyrosine kinases, which are constitutively associated with gp130, leading to the induction of two major signal transduction pathways through the cytoplasmic domain of gp130: the SHP-2/GAB-mediated ERK MAPK pathway and the STAT3-mediated pathway [14,15]. The former is dependent on tyrosine 759 (Y759) of gp130 and the latter requires any one of Y767, Y814, Y904, and Y915, which are all in the YXXQ motif context. An in vivo study using knock-in mice expressing a mutant gp130 defective in either the Y759-dependent signal (SHP-2 signal) or the YXXQ-dependent signals (STAT3 signals) clearly showed that the Y759-mediated signal is required for gp130-mediated ERK MAPK activation, but not for mouse survival, whereas the YXXQ-dependent signals, most likely the STAT3 signals, are required for mouse neonate survival [16]. In addition, the Y759-dependent signal is involved in the defense response against Listeria monocytogenes infection [17], while the YXXQ-dependent signal is required for the acute phase reaction, IgG2a and IgG2b production, Th1 type cytokine production, and B-cell differentiation [16]. The most interesting finding is that Y759 negatively regulates gp130-mediated signals, in particular the STAT3-mediated signals. These results indicate that the balance between a variety of signals generated through a given cytokine receptor, for example gp130, is critical to the determination of the final biological output generated by a given cytokine [14,18]. This concept may be important in considering the mechanisms by which a given cytokine plays a role in health and disease. The first suggestion that IL-6 is involved in autoimmunity came from the findings that cardiac myxoma cells produce IL-6 and that patients with cardiac myxoma frequently show autoimmune symptoms [19]. Furthermore, synovial fluids obtained from patients with rheumatoid arthritis (RA) were found to contain elevated amounts of IL-6 [20]. Since then, several pieces of evidence have been reported that suggest the involvement of IL-6 in autoimmune diseases, chronic inflammatory proliferative disease (CIPD), and B-cell malignancy, including systemic lupus erythematosus (SLE), Castleman’s disease, and plasmacytoma/multiple myeloma [3]. Furthermore, IL-6 is required for experimentally induced autoimmune diseases and autoimmunity, including type II collagen- and antigen-induced arthritis (CIA and AIA) [21–23], myelin oligodendrocyte protein-induced experimental autoimmune encephalomyelitis (EAE) [24,25], and pristane-induced autoantibody production [26]. These results, together with accumulating evidence obtained from a large body of clinical studies, suggest that IL-6-dependent signaling pathways are involved in the pathogenesis of these experimentally induced autoimmune diseases as well as of naturally occurring autoimmune diseases, although it is

unknown how and at what levels IL-6 plays its roles in the complex processes of these diseases. Here we discuss the possible roles of IL-6 in autoimmune diseases and CIPD.

2. IL-6 and chronic inflammation: its implication in polyclonal B-cell activation and autoantibody production Cardiac myxoma is a benign intraatrial heart tumor, and one-third of the patients with cardiac myxoma show autoimmune symptoms such as hypergammaglobulinemia and the production of autoantibodies [19]. These symptoms disappear upon surgical removal of the tumor, suggesting that the myxoma itself or its products are involved in the autoimmune condition of these patients. In fact, culture supernatants of these tumor cells were found to contain high IL-6 activity, and IL-6 mRNA was detected in myxoma cells. Before obtaining this result, we had already found that the pleural effusion cells of patients with pulmonary tuberculosis produce high levels of factors capable of inducing immunoglobulin production [27]. One of these active factors was partially purified and designated as TRF-like factor/IL-6 [2]. Notably, patients with pulmonary tuberculosis often express a wide range of autoantibodies [28], and in certain cases, a diffuse hypergammaglobulinemia has been observed in these patients [29]. These facts suggest that IL-6 is important in the B-cell abnormalities associated with the inflammatory process. Consistent with this idea, inflammation has been implicated in polyclonal B-cell activation and monoclonal B-cell neoplasia. Patients with pre-existing chronic inflammations show polyclonal hypergammaglobulinemia and frequently develop plasma cell neoplasias or lymphoma [28,30–32]. Mineral oil or pristane, which induces chronic inflammation, elicits not only an erosive arthritis resembling RA but also plasmacytoma in certain strains of mice [33–35]. Furthermore, pristane induces a production of autoantibodies characteristic of SLE in BALB/c mice [36]. A role for IL-6 in pristane-induced polyclonal B-cell activation is suggested by the fact that pristane is a strong inducer of IL-6 [37]. Among the autoantibodies produced in response to pristane, IgG anti-DNA and -chromatin autoantibodies are totally IL-6-dependent, whereas IgG anti-nRNP/Sm and -Su antibodies are not [38]. Furthermore, pristane can induce anti-chromatin antibodies in BALB/c nu/nu mice, indicating that production of a subset of autoantibodies by B cells is IL-6-dependent and thymus-independent [26]. Taken together, all these results suggest that chronic inflammation-induced IL-6 production is intimately related to polyclonal B-cell activation and autoantibody production. The involvement of IL-6 in autoantibody production has been further supported by studies of both patients and animal models. SLE is a systemic autoimmune disease characterized by hypergammaglobulinemia, production of a variety of autoantibodies, and glomerulonephritis. Although serum IL-6

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levels do not increase in most SLE patients, elevation of serum IL-6 levels accompanying increased C-reactive protein levels is observed in SLE patients with serositis, with infection, and during disease exacerbations [39–41]. IL-6 is also elevated in the cerebrospinal fluid of patients with central nervous system (CNS) lupus, but not in lupus patients without CNS involvement [42]. Because polyclonal B-cell activation is thought to be a principal immunological abnormality in SLE, a deregulated IL-6/IL-6R system in B cells has been suggested as the underlying cause. B cells from SLE patients spontaneously produce large amounts of IgGs, including IgG anti-ssDNA antibody, in the absence of T cells [43]. Low-density, i.e. activated B cells respond to IL-6 to produce IgGs and IgG anti-ssDNA antibody [43,44]. Abnormal production of IL-6 and expression of the IL-6R in B cells have been shown in SLE and several other autoimmune diseases. Although normal T and B cells do not produce IL-6, SLE B cells, especially high-density B cells, produce greater amounts of IL-6 in culture supernatants than do SLE T cells, regardless of the activity of the disease [43]. In healthy adults, activated B cells, but not resting B cells, express IL-6R [45]. In patients with active SLE, IL-6R and IL-2R are preferentially expressed on low-density B cells [44]. These observations suggest that the polyclonal B-cell activation pathway is affected in patients with SLE: high-density B cells produce IL-6, which activates the low-density B cells, leading to the production of immunoglobulins [46,47]. An anti-IL-6R mAb inhibits the spontaneous production of anti-DNA autoantibodies, indicating that B cells are activated through an IL-6R-mediated signal in SLE [44]. Results from murine systems also support the idea that IL-6 is involved in autoimmune diseases. IL-6 in the sera of MRL/lpr mice increases in an age-dependent manner. This increase is not seen in normal mouse strains, such as BALB/c and C3H/HeN mice or in autoimmune-prone strains, such as (NZB/NZW)F1, and (NZB/BXSB)F1 mice [48]. The increased serum IL-6 in the autoimmune mice does not result from a mutation in the IL-6 gene, because no restriction fragment length polymorphisms in the IL-6 gene of the autoimmune strains NZB, NZW, MRL-lpr/lpr, or BXSB could be detected [49]. IL-6 activity is detected in the sera of MRL/lpr mice as young as 3 weeks of age, and the expression of IL-6 mRNA is detected in the spleen and lymph nodes starting at 8 postnatal weeks, suggesting that increased serum IL-6 is one of the earliest abnormalities to appear in MRL/lpr mice. Expanded abnormal T cells as well as phenotypically normal CD4+ and CD8+ T cells from these mice express IL-6 [50]. Constitutive expression of the IL-6 receptor has also been demonstrated in splenic B cells from aged MRL/lpr mice [51]. Treatment of MRL/lpr mice with an anti-IL-6R antibody reduces anti-dsDNA antibody levels and ameliorates the nephritis, suggesting that abnormal IL-6 and IL-6R systems are involved in the pathophysiology of the autoimmune disease of MRL/lpr mice [52]. Although an increase in serum IL-6 levels is not observed in NZB/W F1 mice, spontaneous production of

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an IgG anti-DNA antibody in vitro by splenic B cells from these mice was enhanced by IL-6, indicating the hyper-responsiveness of these B cells to IL-6. The ability of B cells to produce IgG anti-DNA antibodies seems to be related to the expression levels of IL-6R at the age when the mice begin to develop the disease [53]. Administration of IL-6 to NZB/W F1 mice results in an accelerated and severe form of membranoproliferative glomerulonephritis [54]. Treatment of NZB/W F1 mice with an anti-IL-6 mAb prevents production of anti-dsDNA, significantly reduces proteinuria, and prolongs life [55]. Similar effects have also been reported for the IL-6 receptor blockade of NZB/W F1 mice [56]. All these results indicate that IL-6/IL-6R systems are involved in the promotion of autoimmune disease in NZB/W F1 mice. In addition to autonomous B-cell abnormalities, AlarconRiquelme et al. [57] proposed that macrophages are involved in autoantibody production in NZB/W F1 mice, because spontaneous production of IL-6 and IgG autoantibodies was reduced by the depletion of macrophages. However, a significant increase in IL-6 production by NZB/W F1 peritoneal exudate cells was not observed in this study. Defective cytokine production, including production of IL-6, is common in macrophages from autoimmune disease prone mice [58,59]. Peritoneal macrophages from several autoimmune disease prone mouse strains (MRL/lpr, MRL/+, NZB/W, BXSB, and B6/gld) show defective expression of the cytokines IL-1␣, IL-1␤, and IL-6, but not TNF␣, before the onset of overt disease. The same defects have been seen in bone marrow-derived macrophage precursors, suggesting that these defects are intrinsic to macrophages and are not a result of conditioning by the autoimmune environment [58]. Furthermore, endotoxin-activated macrophages from MRL+/+ and NZB/W F1 mice, obtained well before the onset of disease signs, have defective TNF␣ production, leading to impaired production of IL-1 and IL-6 [59]. These findings suggest that impaired pro-inflammatory cytokine production by macrophages may be associated with systemic autoimmunity, and that the main causes of polyclonal B-cell activation in NZB/W F1 mice seem to be intrinsic B-cell abnormalities, hyper-responsiveness and IL-6 production. Another interesting piece of evidence is that a striking increase in the level of agalactosyl IgG has been observed in a variety of autoimmune- and/or IL-6-related diseases, such as pulmonary tuberculosis, RA, Crohn’s disease (CD), sarcoidosis, leprosy, Castleman’s disease, Takayasu’s arteritis, multiple myeloma, and MRL/lpr mouse and pristane-induced arthritis [32,60–62]. Furthermore, IL-6 transgenic mice show a marked increase in agalactosyl IgG, suggesting that IL-6 may directly increase the level of agalactosyl IgG [60]. Such evidence further strengthens the suggestion of an intimate relationship between IL-6 and certain autoimmune and/or idiopathic diseases. Furthermore, agalactosyl IgG has an enhanced ability to interact with lectin mannose-binding protein (MBP), an acute phase protein that is inducible by IL-6, which can trigger the

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complement cascade through MBP [63]. There are several reports indicating a correlation between low serum MBP levels with CRP, IgM-rheumatoid factor (RF) [64], or poor prognosis for RA [65,66].

3. Overexpression of IL-6 results in the generation of polyclonal plasmacytosis and malignant plasmacytoma Mice expressing IL-6 as a transgene (IL-6 transgenic mice) develop a massive polyclonal plasmacytosis with autoantibody production and mesangial cell proliferative glomerulonephritis that resembles the autoimmune diseases observed in NZB/W F1 mice or SLE patients [67]. The development of hypergammaglobulinemia has also been reported in mice whose bone marrow has been altered by the transplantation of cells infected with a retroviral vector expressing murine IL-6 [68]. Plasma cells generated in IL-6 transgenic mice were not transplantable to syngeneic animals, indicating that additional factors may be required for malignant transformation [67]. In this context, it is interesting to consider the fact that susceptibility to pristane-induced plasmacytomagenesis is genetically determined and that most inbred strains other than BALB/c are resistant [69]. Consistent with this observation, C57BL/6 IL-6 transgenic mice show a progression from polyclonal plasmacytosis to fully transformed monoclonal plasmacytoma when backcrossed to BALB/c mice. These mice display chromosomal translocation with c-myc gene rearrangement [70]. The evidence strongly supports the hypothesis that deregulated expression of the IL-6 gene can trigger polyclonal plasmacytosis resulting in the generation of a malignant monoclonal plasmacytoma [71]. Consistent with this idea, IL-6-deficient mice do not develop plasmacytoma [72], indicating the critical role played by IL-6 in murine plasmacytomagenesis.

4. Insulin-dependent diabetes mellitus and IL-6 Several pieces of evidence support a role for IL-6 in the diabetes developed by the nonobese diabetic (NOD) mouse, an animal model for insulin-dependent diabetes mellitus (IDDM). IL-6 production is first detected in the pancreas at 10 weeks of age and disappears by 16 weeks in both NOD and BALB/c mice. It is also present in the endothelial cells [73]. IL-6 production by the cells that infiltrate the Langerhans’ islets and in the endocrine islet of NOD females is found at all ages [74]. ␤-cell destructive insulitis in NOD mice is associated with increased expression of pro-inflammatory cytokines (IL-1, TNF␣, and IFN␣) and type 1 cytokines (IFN␥, TNF␤, IL-2, and IL-12). Postnatal deletion of IL-6 by systemic administration of neutralizing antibodies significantly decreases the incidence of IDDM in NOD mice [75] and/or BB rats, another IDDM model, indicating that there are pathologic roles for IL-6 as for other

pro-inflammatory cytokines (IL-1, TNF␣) and type 1 cytokines (IFN␥, IL-2, and IL-12) in IDDM development [76]. The involvement of IL-6 in autoimmune insulin-dependent type I diabetes is further suggested by results from IL-6 transgenic mice, which overexpress IL-6 in the pancreatic islet ␤-cells [77]. Although these transgenic mice remain normoglycemic throughout their lives, histopathological examination reveals that, particularly in older mice, there is a florid insulitis that is composed predominantly of B220+ B lymphocytes and, to a lesser extent, Mac-1+ macrophages and T lymphocytes. Furthermore, infiltration of plasma cells into the peri-islet is observed. These facts support the idea that the overexpression of IL-6 induces B-cell differentiation, which may be important in the development of autoimmune disease. This concept is supported by the fact that anti-IL-6 antibodies inhibit the development of insulin-dependent diabetes in NOD/Wehi mice [75]. Unexpectedly, a critical role for B cells in type 1 diabetes was recently evidenced by the protection of B-cell-deficient NOD mice (NOD ␮MT) from this form of diabetes [78]. In humans, a novel gene, encoding hepatocarcinomaintestine-pancreas/pancreatic-associated protein (HIP/PAP), is reportedly overexpressed in the pancreatic islets of patients with new-onset type 1 diabetes; this protein is a potential candidate for novel disease-related autoantigens. Interestingly, this gene contains a putative IL-6 response element, and release of the HIP/PAP protein from islets into the culture medium is enhanced by the addition of IL-6 [79].

5. Inflammatory bowel disease and IL-6 Serum IL-6 concentrations are significantly higher in patients with Crohn’s disease (CD) than in patients with ulcerative colitis (UC) and healthy controls [80,81]. In individual patients, serum IL-6 levels correlate with the corresponding CD activity index in patients with a primarily inflammatory disease and without bowel stenosis, previous intestinal resection, or concomitant inflammatory disorders [82]. Longitudinally measured serum IL-6 levels reflect the patients’ clinical response during steroid therapy and predict clinical relapse after steroid-induced remission at week 9 of the treatment protocol [82]. In CD, the persistent elevation in serum soluble IL-2R levels during remission corresponds to chronic active disease, while high serum levels of IL-6 in these patients are associated with a high frequency of relapse [83]. These studies indicate that serum IL-6 is a clinically relevant parameter for CD that correlates with inflammatory activity and implies a prognostic value after steroid-induced remission. To find sources of IL-6 production, peripheral blood and specimens from endoscopic biopsy have been examined. In an earlier study, an increase in spontaneous as well as inducible IL-6 production by peripheral blood mononuclear leukocytes from patients with CD was observed [84,85]. The increased serum levels and synthesis of IL-6 by peripheral

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blood mononuclear cells are accompanied by IL-1 and TNF␣ production [81], reflecting the inflammatory state of the patient. Analysis of endoscopic biopsy samples from IBD patients revealed that the involved colonic mucosa from patients with active disease contains larger amounts of IL-6 than colonic mucosa from patients with inactive disease or normal controls [86]. RT-PCR analysis showed that, of the pro-inflammatory cytokines tested, IL-6 mRNA levels are highest in patients with active IBD [87]. Infiltrating T cells, macrophages, and B cells were identified as sources of IL-6 protein in IBD specimens by immunofluorescent staining [87]. IL-6 transcripts are elevated only in active IBD specimens, suggesting that IL-6 is involved in ongoing immune processes in the inflammatory mucosal environment. IL-6 may play roles not only in mediating inflammation but also in the pathogenesis of IBD, because there are IBD cases where IL-6 is present in enterocytes and colocytes in both inflamed and non-inflamed small and large intestines, with no IL-6 in the infiltrating inflammatory cells of the lamina propria (LP) [88]. An analysis of cytokine production by lamina propria mononuclear cells (LPMNC) revealed unique characteristics of UC. LPMNC isolated from involved IBD (UC and CD) mucosa spontaneously produces increased amounts of TNF␣, IL-6, and IL-1␤. The secretion patterns of TNF␣ and IL-1␤ by LPMNC from patients with either UC or CD demonstrate a close correlation with the degree of tissue involvement and mucosal inflammation. LPMNC from non-involved UC mucosa secretes markedly increased levels of IL-6 compared with non-involved CD mucosa or control mucosa. The heightened IL-6 secretion from LPMNC from non-involved UC mucosa without visible or microscopic signs of inflammation indicates that the pathophysiologic mechanisms involved in the initiation of inflammation may differ between UC and CD [89]. Although increased production of the macrophage-derived cytokines TNF␣, IL-1, and IL-6 in both UC and CD has been reported, there are specific changes in CD but not UC: in active CD, large numbers of activated T lymphocytes secreting IL-2 and IFN␥ can be detected in the lamina propria [90]. This suggests considerable involvement of immune deregulation in the pathogenesis of CD, and IL-6 could be a candidate because its increase in the serum is specific for CD. IL-6 can bind to cells lacking the IL-6 receptor when it forms a complex with the soluble IL-6R (receptor conversion/trans-signaling). The soluble IL-6R and receptor conversion/trans-signaling have an essential pathogenic function in autoimmune disease models and IBD [91]. Most LP T cells from CD lack the IL-6R but produce large amounts of IL-6 in vitro and their STAT3 is activated. LP T cells express the STAT3 target genes Bcl-2 and Bcl-XL and are resistant to apoptosis. Anti-IL-6R antibody inhibits the activation of STAT3 and induces the apoptosis of LP T cells, indicating that the activation of LP T cells as well as their resistance to apoptosis is IL-6R dependent. Because stimulated LP mononuclear cells produce soluble IL-6R,

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the LP T cells that lack IL-6R could be activated by the trans-signaling/receptor conversion. This hypothesis was confirmed in vivo using several experimental models of IBD. Administration of an anti-IL-6R antibody inhibits the activation of STAT3 of the spleen, and wasting disease, rectal prolapse, and macroscopic as well as histological inflammation of the intestine are prevented in colitis in SCID mice adoptively transferred with CD62L+ CD45RBhigh CD4+ T cells. In the TNBS-induced colitis model, anti-IL-6R antibody as well as gp130Fc significantly reduces the severity of the colitis. Apoptosis of spleen cells in vitro is induced by the blockade of the IL-6/IL-6R system by the anti-IL-6R antibody as well as by gp130Fc. Histologically, apoptosis of CD4+ T cells in the colon caused by the blockade of the IL-6/IL-6R system has been demonstrated. These data indicate that a pathway of T-cell activation driven by IL-6-sIL-6R contributes to the perpetuation of chronic intestinal inflammation [91]. Suzuki et al. examined the molecular mechanisms for gp130-mediated signals in IBD, the involvement of the STAT transcription factors in IBD. They found that among the STAT family members, STAT3 is most strongly tyrosine phosphorylated in human UC and CD patients and in dextran sulfate sodium (DSS)-induced colitis in mice [92]. The development of colitis as well as STAT3 activation is significantly reduced in IL-6-deficient mice treated with DSS, suggesting that IL-6-mediated STAT3 activation plays an important role in the perpetuation of colitis [92]. Suzuki et al. also examined the roles for suppressor of cytokine signaling (SOCS) families in IBD. Although both SOCS1/JAB (JAK-binding protein) and SOCS3 are potent inhibitors of the biological effects mediated by the IL-6 family cytokines, their inhibitory mechanisms are distinct. SOCS3, but not SOCS1/JAB, is highly expressed in the colon of DSS-treated mice as well as several T-cell-dependent colitis models. To understand the roles of SOCS in IBD, transgenic mice expressing a JAB mutant (F59D-JAB) that overcomes the inhibitory effect of both JAB and SOCS3 were created. DSS induced stronger STAT3 activation and more severe colitis in the F59D-JAB transgenic mice than in their wild-type littermates, indicating that SOCS3 plays a negative regulatory role in intestinal inflammation by down-regulating STAT3 activity [92].

6. Experimental autoimmune encephalomyelitis and IL-6 Experimental autoimmune encephalomyelitis is induced by immunization with myelin components such as myelin oligodendrocyte glycoprotein (MOG) and is used as an animal model for a demyelinating disease, multiple sclerosis. Myelin-specific Th1 cells enter the CNS via the binding of very late antigen 4 (VLA-4), which they express, to the endothelial vascular cell adhesion molecule 1 (VCAM-1). IL-6-deficient mice are resistant to the MOG-induced EAE,

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compared with wild-type mice [24,25,93]. The delayed-type hypersensitivity response, lymphocyte proliferation response, and antibody reactivity to MOG in IL-6-deficient mice are significantly lower than in wild-type mice. The resistance to EAE in IL-6-deficient mice is associated with a deficiency of MOG-specific T cells, which can differentiate into either Th1 or Th2 type effecter cells in vivo [25]. Histologically, no infiltration of inflammatory cells is observed in the CNS of IL-6-deficient mice. This is due to the decreased expression of endothelial VCAM-1 and ICAM-1 in MOG-primed IL-6-deficient mice, which is up-regulated in the CNS in wild-type mice [24]. IL-6 may play a crucial role in the induction phase of EAE, because treatment of IL-6-deficient mice with IL-6 during the pre-clinical phase and the passive transfer of lymphocytes from wild-type mice could induce EAE in IL-6-deficient mice [94]. Further analyses are required to understand the mechanisms underlying how IL-6 regulates the immune responses in EAE.

7. Rheumatoid arthritis and IL-6 RA is a heterogeneous, chronic joint disease that is characterized by leukocyte invasion and synoviocyte activation followed by cartilage and bone destruction [95]. It has properties of both autoimmune and chronic proliferative inflammatory diseases [3]. In the 1970s, B cells and their products were the preferred candidate effecter cells for RA [96]. The evidence supporting this idea was that RA is frequently associated with polyclonal B-cell activation, the production of autoantibodies, and the localization of immune complexes to the joint [97]. More recently, the emphasis has changed to T cells as the likely key player in RA, because of the association of HLA-DR with the disease, which has led to the idea that CD4+ T cells are likely to play a central role in the initiation and progression of RA-associated joint disease [98]. A central role for macrophages or dendritic cells has also been proposed, based on the observation that pro-inflammatory cytokines, such as IL-1, IL-6, and TNF are produced abundantly in the joint [3,95], and on the strong evidence that anti-TNF␣ antibody successfully suppresses the disease of RA patients [95,99,100]. The possible involvement of IL-6 in RA was first demonstrated by the high levels of IL-6 detected in synovial fluid from the joints of patients with active RA [20]. Abnormal IL-6 production is observed in patients with RA where B cells are activated and produce a variety of autoantibodies, such as RF, which is an IgG antibody against the Fc portion of IgG [20,101,102]. There is also a significant correlation between serum IL-6 activity and the serum levels of a variety of acute phase proteins [101] and between the concentrations of synovial fluid IL-6 and the levels of IgG, IgM-RF, or IgG-RF in RA [103,104]. The involvement of IL-6 in the pathophysiology of another arthritic disease, juvenile chronic arthritis (JCA), has been suggested. Patients with systemic-onset JCA have significantly elevated serum IL-6

levels during active disease [105]. Serum IL-6 levels correlate with the extent and severity of joint involvement [105]. In the synovial fluids, high IL-6 levels are found in patients with JCA with polyarticular onset of disease, and the IL-6 levels correlate with the levels of synovial IgM RF [106]. IL-6 production is also observed in type II CIA in mice [107] and MRL/lpr mice [48], which develop autoimmune disease with proliferative glomerulonephritis and arthritis. These results suggest that IL-6 and RF are produced within the rheumatoid joints as a result of abnormal immune system activation, which is associated with the disease activity of RA. Many cell types, including macrophages, T, B, and endothelial cells, fibroblasts, synoviocytes, and chondrocytes can produce IL-6 [20,108,109]. The main cell types producing IL-6 of cells dissociated from RA synovial tissue are mononuclear leukocytes. In situ hybridization of IL-6 mRNA showed positive cells both in the lymphocyte-rich aggregates and adjacent to small vessels [110]. The cells containing the IL-6 mRNA were T cells in contact with CD14+ tissue macrophages [110]. RA fibroblast-like synoviocytes produce IL-6 and IL-8, which contribute to inflammation and joint damage. Because the proliferation of synoviocytes is characteristic of the pathological changes of RA, IL-6 production by them has pivotal effects in the joint. Thus, several factors that regulate the production of IL-6 by RA fibroblasts have been identified. TNF␣, IL-1␤, and IL-17 induce IL-6 production by synovial fibroblasts [108,110,111]. Glucocorticoids strongly, and PGE2 slightly inhibit IL-1-induced IL-6 mRNA expression in synoviocytes [110]. TGF-␤ increases the production of IL-6. CCL2/monocyte chemotactic protein-1, CCL5/RANTES, and CXCL12/stromal cell-derived factor-1 also enhance IL-6 production by fibroblast-like synoviocytes from patients with RA [112]. In RA fibroblasts, calcitonin gene-related peptide (CGRP) increases IL-6 and IL-8 secretion, which is not observed in fibroblasts from osteoarthritis patients. The extent of IL-6 and IL-8 production by RA fibroblasts in response to TNF␣ is greatly augmented by thioredoxin, which accumulates in the synovial fluid of RA, by augmenting the NF-kB activation pathway [113]. NF-kB is essential for IL-6 expression, because fibroblasts in which both NF-kB p50/p65 genes are deleted fail to express IL-6 in response to IL-1 [114]. Because IL-6 has stimulatory effects in osteoclasts, it may inhibit bone formation and induce bone resorption [115,116]. Using IL-6-deficient mice, several groups have examined the roles for IL-6 in the pathophysiology of experimentally induced autoimmune diseases or autoimmunity. IL-6 deficiency results in delay of onset and reduced severity of type II-CIA [21,22]. Alonzi et al. reported that IL-6 is essential for the development of CIA, which they showed using IL-6-deficient mice that had been backcrossed into DBA1/J for five generations. However, an analysis by Sasai et al., using IL-6-deficient mice that had been backcrossed into DBA1/J for eight generations, revealed that IL-6 is not essential for the development of CIA, but is related to the onset

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and severity of arthritis. The humoral and cellular responses to type II collagen of the IL-6-deficient mice were reduced to about half those of wild-type mice. In addition, the helper T-cell responses of IL-6-deficient mice were shifted to the Th2 type, as indicated by the enhanced production of IL-4 and IL-10 in response to concanavalin A stimulation [22]. These results suggest that IL-6 at least regulates the intensity of the immune responses induced by type II collagen. Antigen-induced arthritis in IL-6-deficient mice is mild, and the articular cartilage is well preserved, whereas it is destroyed completely in wild-type mice [23]. In addition, comparable mRNA expression for both IL-1␤ and TNF␣, but not for IL-6, is detected in the inflamed joints of IL-6-deficient mice, suggesting that IL-6 may play a crucial role in cartilage destruction. Both the antigen-specific in vitro proliferative response in lymph node cells and the in vivo antibody production elicited in the IL-6-deficient mice are reduced to less than half of those seen in wild-type mice. The lymph node cells of IL-6-deficient mice produce much more Th2 cytokines than lymph node cells in wild-type mice with either antigen-specific or non-specific stimulation in in vitro culture. These results indicate that IL-6 plays a key role in the development of AIA at the inductive as well as the effecter phase. To clarify the roles for IL-6 in murine arthritis models, other model systems have been examined using the IL-6-deficient mice. Passive immune complex-induced arthritis does not differ between wild-type and IL-6-deficient mice. Non-immunologically mediated zymosan-induced arthritis develops similarly in the first week, but only wild-type mice develop chronic synovitis. These results indicate an important role for IL-6 in propagation of joint inflammation, potentially independent of its role in immunity [117]. On the other hand, arthritis in TNF␣ transgenic mice is not affected by the inactivation of the IL-6 gene [21]. Considering that the arthritis of TNF∆ARE mice, which also exhibit an increase in serum TNF␣, does not require lymphocytes, IL-6 but not TNF␣ seems to have immuno-regulatory roles in arthritis. Furthermore, heterogeneity of the molecular mechanisms of arthritis in murine models is also suggested by these results, as is the case for human RA.

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pseudogene localized to chromosome 17. The murine and rat gp130 genes are located on chromosome 13 and 2q14-q16, respectively. There are four polymorphisms in the IL-6 promoter (−597G → A, −572G → C, −373A(n)T(n), −174G → C), which are naturally occurring haplotypes. A polymorphism in the 5 flanking region of the IL-6 gene at position –174 (G → C) appears to affect IL-6 transcription. After coronary artery bypass surgery, patients with the −174C/C allele show higher plasma IL-6 levels than patients with the −174G/G allele [118]. In addition, a lower frequency of the −174C/C genotype is observed in JCA patients than in the normal population [119]. The C/G variation at position −174 is also associated with susceptibility to type-I diabetes mellitus [120], but no association is observed with systemic lupus erythematosus in Caucasian German patients [121] or in IBD patients [122]. However, another in vitro study suggested that genetic polymorphisms in the promoter influence IL-6 transcription through complex interactions determined by the haplotype. For example, the transcription of IL-6 is higher from the GG9/11G haplotype and lower from the AG8/12G allele [123]. The AT-rich minisatellite allele distribution pattern is significantly different in SLE compared with controls in Caucasian and African–American patients. In both races, short allele sizes (less than or equal to 792 bp) were seen exclusively in SLE patients, whereas the 828 bp allele was predominant in controls. A biological significance for 3 minisatellite alleles is suggested by the higher IL-6 secretion by B lymphoblastoid cells, higher percentages of IL-6 positive cells, and enhanced stability of IL-6 mRNA in patients with SLE [121]. In Sardinian simplex families with multiple sclerosis, nine alleles have been identified in a minisatellite polymorphism located in the 3 flanking region of the IL-6 gene. Of these, the A5 allele, especially the A5/A5 genotype, is associated with a benign course of disease. In addition, the presence of any of the larger alleles (A6 → A9) is associated with accelerated onset of disease [124]. Another polymorphism in the forth intron (G to A at position 4470) appears to correlate with CD and UC although no significant alteration of IL-6 expression is associated with this polymorphism [125].

8. Polymorphisms in the human IL-6 gene 9. Possible mechanisms As described above, because several lines of evidence support the involvement of IL-6 in various autoimmune diseases, several groups have intensively examined the genetic polymorphisms related to these diseases. The human IL-6 gene is located on chromosome 7p21. The chromosome localizations of the IL-6 gene of mouse and rat are chromosomes 5 and 4, respectively. The human IL-6R␣ gene is located on chromosome 1q21. The mouse and rat IL-6R␣ genes are on chromosomes 3 and 2, respectively. The human has two chromosomal loci for gp130; one is the genuine gp130 gene, located on chromosome 5q11, and the other is a

Because STAT3 activation is one of the prominent features of IL-6 stimulation, the activation state of STATs in samples of arthritic joints from patients or experimental models has been examined. STAT3 is strongly tyrosine phosphorylated in the synovial tissue of RA patients, but not of patients with osteoarthritis [126]. Furthermore, the mRNA for the endogenous cytokine signaling repressor CIS3/SOCS3 is abundantly expressed in RA patients. In murine experimental arthritis models, Shouda et al. observed tyrosine phosphorylation of STAT3 in the ankle joints around day

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40 in the course of CIA. Induction of SOCS3 was delayed until around day 50 and was observed in the ankles of mice with AIA. To understand the role of STAT3 and SOCS3 in arthritis, Shouda and colleagues injected a recombinant adenovirus carrying dominant-negative STAT3 or wild-type SOCS3 cDNA periarticularly into the ankle joints of mice with AIA or CIA. Periarticular injection of dnSTAT3 or SOCS3 adenovirus reduced the severity of arthritis and joint swelling compared with controls. SOCS3 was more effective than the dominant-negative form of STAT3 in the CIA model. These experiments clearly demonstrate that the effects of IL-6 in these experimental arthritis models are mediated by the activation of STAT3. Furthermore, they suggest that blockade of the IL-6-gp130-JAK-STAT3 signaling pathway and induction of SOCS3 may be beneficial in the treatment of RA [126]. These results suggest that IL-6-dependent signaling pathways are involved in the pathogenesis of these experimentally induced autoimmune diseases. Because IL-6 belongs to the family of pro-inflammatory cytokines, it can be readily appreciated that IL-6 plays roles in the chronic inflammation that is caused by autoimmunity. However, it is unknown whether IL-6 and its receptor system are involved in the pathogenesis of spontaneously occurring autoimmune diseases. An important question is whether the IL-6/IL-6 receptor system is involved in the regulation of thymic selection or maintenance of self-tolerance. The reduction of T cells without accompanying developmental defects in the thymus and periphery of IL-6 knock-out mice indicates that IL-6 is important in the maintenance of levels of T-cell numbers rather than in development. There are several reports indicating a role for IL-6 in supporting the survival of T cells. IL-6 supports the survival of na¨ıve T cells in vitro [127]. STAT3 is involved in the regulation of survival and activation of T cells [128,129]. The anti-apoptotic effects of IL-6 on na¨ıve T cells ex vivo are reportedly mediated by the induction of STAT1 activation selectively in na¨ıve but not activated/memory T cells [130]. In addition to the anti-apoptotic effects of IL-6 on na¨ıve T cells, the inhibition of activation-induced cell death and down-regulation of FasL by IL-6 have been reported in the case of a T-cell hybridoma [131]. There have been no analyses to answer whether IL-6 is involved in the formation of lymphocyte repertoire, probably because no mutant mice related to IL-6 signals have exhibited the phenotypes suggesting the impairment of self-/non-self-recognition. In the pathogenesis of autoimmune disease, the breakdown of self-tolerance is a critical event. In the thymus, central tolerance is generated during T-cell development to remove self-reactive T cells and is involved in the primary repertoire formation. Peripheral tolerance is induced after the antigen-specific responses. Because T cells recognize antigens expressed by antigen-presenting cells (APCs) in the context of self-MHC molecules, it is reasonable to think that changing APC function may cause autoimmune diseases. It has been reported that IL-6 affects the antigen processing of bone marrow-derived dendritic cells (BMDCs) so that

cryptic epitopes are presented [132]. Murine BMDCs were loaded with the antigen hen-egg lysozyme (HEL) in the presence or absence of IL-6, and the antigen presentation of the BMDCs was detected by the IL-2 production of a panel of T-cell hybridomas specific to HEL-derived peptide sequences. The hybridoma specific to HEL peptide 2-16 (HEL 2-16) usually does not respond to BMDCs loaded with HEL, because peptide 2-16 is not presented by BMDCs. This means that peptide 2-16 has a cryptic epitope. When HEL was loaded into BMDCs in the presence of IL-6, the hybridoma HEL 2-16 was stimulated by the BMDCs, indicating that the IL-6 treatment of BMDCs resulted in the presentation of a cryptic epitope. Treatment of BMDCs with IL-6 neither induces maturation, such as by altering the expression levels of class II MHC and co-stimulatory molecules, nor changes the rate of endocytosis. The critical effect of IL-6 in modifying the quality of peptide presentation seems to be the acidification of endosomes. IL-6 acidifies early endosomes located in the periphery 10 min after antigen loading. The pH of the early endosomes is controlled by the membrane Na+ /K+ ATPase, and inhibition of this protein pump acidifies early endosomes. Inhibition of this pump by IL-6 in hepatocytes has been reported [133]. The fact that chloroquin, a classical inhibitor of this pump, induces the presentation of HEL 2-16 by BMDCs in the absence of IL-6 supports the possibility that acidification of early endosomes by IL-6 causes the presentation of cryptic epitopes. It is intriguing to speculate that the dysfunction of the Na+ /K+ ATPase in the mononuclear cells from RA patients [134] is mediated by increased serum IL-6. Thus, it is important to clarify the signaling pathway and molecular mechanisms by which IL-6 regulates the Na+ /K+ ATPase.

10. Conclusion and future prospects IL-6 is a pleiotropic cytokine originally identified as a B-cell differentiation factor. Its excess activity in vivo results in polyclonal B-cell activation, plasmacytosis, and B-cell neoplasia. These changes provide a basis for autoantibody production. At the beginning of acute inflammation, IL-6 mediates the acute phase responses. When its activity as a pro-inflammatory cytokine persists, acute inflammation turns into chronic inflammation that includes immune responses. In the chronic phase of inflammation, continuous signals provided from IL-6 support the survival and growth of lymphocytes and myeloid cells, which may increase the serum IL-6 levels. This situation provides the basis for the amplification step of chronic inflammatory proliferation. Plasmacytosis and hyperplasia of synovial cells in the joints of RA patients are a typical example of chronic inflammatory proliferation. In autoimmune diseases, IL-6 not only maintains inflammation, but also modifies the immune responses. One example has suggested that IL-6 can change the direction of immune responses to self or non-self, or activation or tolerance, by modifying the quality of peptide

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presentation by BMDCs. It is not completely understood if there are other ways for IL-6 to modify immune responses. Autoimmune diseases are thought to be polygenic diseases. It is possible that IL-6, IL-6R␣, or gp130 is involved in autoimmune diseases. We still need to clarify the molecular mechanisms of IL-6-mediated autoimmune diseases, using a series of knock-out/knock-in mice for IL-6R-related genes.

Acknowledgements We thank our colleagues, who contributed to our recent studies described in this review. We also thank Ms. R. Masuda and A. Kubota for secretarial assistance. This work is supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology in Japan, and the Osaka Foundation for the Promotion of Clinical Immunology.

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