Miho Suzuki

IL-6/IL-6 receptor system and its role in physiological and pathological conditions

IL (interleukin)-6, which was originally identified as a B-cell differentiation factor, is a multifunctional cytokine that regulates the immune response, haemopoiesis, the acute phase response and inflammation. IL-6 is produced by various types of cell and influences various cell types, and has multiple biological activities through its unique receptor system. IL-6 exerts its biological activities through two molecules: IL-6R (IL-6 receptor) and gp130. When IL-6 binds to mIL-6R (membrane-bound form of IL-6R), homodimerization of gp130 is induced and a high-affinity functional receptor complex of IL-6, IL-6R and gp130 is formed. Interestingly, sIL-6R (soluble form of IL-6R) also binds with IL-6, and the IL-6-sIL-6R complex can then form a complex with gp130. The homodimerization of receptor complex activates JAKs (Janus kinases) that then phosphorylate tyrosine residues in the cytoplasmic domain of gp130. The gp130-mediated JAK activation by IL-6 triggers two main signalling pathways: the gp130 Tyr759-derived SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase-2)/ERK (extracellular-signal-regulated kinase) MAPK (mitogen-activated protein kinase) pathway and the gp130 YXXQ-mediated JAK/STAT (signal transducer and activator of transcription) pathway. Increased IL-6 levels are observed in several human inflammatory diseases, such as rheumatoid arthritis, Castlemans disease and systemic juvenile idiopathic arthritis. IL-6 is also critically involved in experimentally induced autoimmune diseases. All clinical findings and animal models suggest that IL-6 plays a number of critical roles in the pathogenesis of autoimmune diseases. In the present review, we first summarize the IL-6/IL-6R system and IL-6 signal transduction, and then go on to discuss the physiological and pathological roles of IL-6.

Overproduced interleukin 6 decreases blood lipid levels via upregulation of very-low-density lipoprotein receptor

Background Interleukin 6 (IL6) blockade raises blood lipid levels in patients with rheumatoid arthritis. Objective To examine the influence of IL6 on lipid metabolism. Methods Vascular smooth muscle cells (VSMC) were cultured in the presence of IL6, soluble IL6 receptor (sIL6R), IL6+sIL6R or tumour necrosis factor α (TNFα) for 24 h. After culture, the expression of very-low-density lipoprotein receptor (VLDLR), low-density lipoprotein receptor (LDLR) and low-density lipoprotein-related protein-1 (LRP-1) were measured by real-time PCR. Human IL6 was injected into mice twice a day for 2 weeks and then VLDLR expression in several tissues and the change of total cholesterol (TC) and triglyceride (TG) levels were investigated. Finally, the effect of anti-IL6 receptor (IL6R) antibody injection on blood lipid levels was examined. Results IL6+sIL6R significantly induced expression of VLDLR mRNA in VSMC (8.6-fold, p<0.05), but IL6 or sIL6R alone and TNFα did not do so. None of these cytokines induced LDLR and LRP-1 mRNA expression. IL6 injection into mice increased the expression of VLDLR in heart, adipose tissue and liver and decreased TC and TG levels. The injection of anti-IL6R antibody normalised the reduced levels of TC and TG caused by IL6 injection, whereas it had no influence on the levels of TC and TG in normal mice. Conclusions Overproduced IL6 decreased blood lipid levels by increasing VLDLR expression in several tissues. It is concluded that IL6 blockade normalises reduced lipid levels caused by IL6, but does not affect normal lipid metabolism.

IL-6 and IL-1 synergistically enhanced the production of MMPs from synovial cells by up-regulating IL-6 production and IL-1 receptor I expression.

In the present study, we investigated potential synergism between IL-6 and IL-1 for the production of matrix metalloproteinases (MMPs) by the synovial cell line SW982. Cells were cultured with different combinations of IL-6, soluble IL-6 receptor (sIL-6R) and IL-1beta for 24h and production of MMPs was then measured. IL-6+sIL-6R, but not IL-6 alone, induced MMP-13 and MMP-3 production. IL-1beta also induced production of MMPs. Of interest, addition of IL-6+sIL-6R together with IL-1beta synergistically increased MMP production. Next, we analyzed the mechanism responsible for the synergistic effects of IL-6+sIL-6R and IL-1beta in combination. IL-1beta-induced MMP production was significantly augmented in the presence of sIL-6R. IL-1beta as well as IL-6+sIL-6R induced IL-6 production. Moreover, IL-6+sIL-6R significantly augmented expression of IL-1RI, but not IL-1RII, in SW982 cells. Responsiveness to IL-1beta was much higher in IL-6+sIL-6R-pretreated cells than non-treated cells in terms of MMP production. Finally, IL-6+sIL-6R-induced IL-1RI expression was inhibited by a STAT pathway inhibitor, but not a MAPK pathway inhibitor. These results suggest that increased expression of IL-1RI stimulated by IL-6+sIL-6R and the increased production of IL-6 on exposure to IL-1beta and IL-6+sIL-6R are involved in the observed synergistic effect on the production of MMPs by SW982 cells.

Anti-interleukin-6 receptor antibody prevents systemic bone mass loss via reducing the number of osteoclast precursors in bone marrow in a collagen-induced arthritis model

Systemic bone loss is a hallmark of rheumatoid arthritis (RA). Inflammatory cytokines such as interleukin (IL)‐6 promote bone resorption by osteoclasts. Sphingosine‐1‐phosphate (S1P) controls the migration of osteoclast precursor cells (OCPs) between the blood and bone marrow, in part via S1P receptors (S1PR1 and S1PR2) expressed on the surface of OCPs. OCPs (CD11b+Gr‐1low+med) isolated from bone marrow of DBA/1J mice were stimulated with IL‐6. S1P‐directed chemotaxis of OCPs was evaluated using a transwell plate. mRNA expression of S1PR1 and S1PR2 was measured. DBA/1J mice were immunized with bovine type II collagen (days 0 and 21) and anti‐mouse IL‐6 receptor antibody (MR16‐1) was administered on days 0 and/or 21. Trabecular bone volume was analysed using micro‐computed tomography. The percentage of OCPs in tibial bone marrow and S1PR1 and S1PR2 mRNA expression in OCPs were measured. IL‐6 stimulation significantly decreased S1P‐directed chemotaxis of OCPs. IL‐6 induced S1PR2 mRNA expression, but not S1PR1 mRNA expression, in OCPs. Bone volume was significantly lower in arthritic mice than in non‐arthritic control mice on day 35. Treatment of immunized mice with MR16‐1 significantly inhibited bone loss. In MR16‐1‐treated mice, the percentage of OCPs and expression of S1PR2 mRNA was each decreased compared with arthritic mice on day 14, but not on day 35. IL‐6 increased the number of OCPs in tibial bone marrow via up‐regulating S1PR2, thus playing a crucial role in systemic bone loss induced by inflammation.

Anti-IL-6 receptor antibody suppressed T cell activation by inhibiting IL-2 production and inducing regulatory T cells

T cell activation is crucial to the pathogenesis and progression of rheumatoid arthritis. Tumour necrosis factor-alpha (TNFalpha) and interleukin (IL)-6 inhibitors show marked efficacy in rheumatoid arthritis patients, but their impacts on T cell activation have remained unclear. To shed light on these impacts, we examined the effects of an anti-IL-6 receptor antibody and an anti-TNFalpha antibody on T cell activation in two experimental systems: spleen cells stimulated by anti-CD3 antibody, and purified splenic CD4 T cells stimulated by both anti-CD3 and anti-CD28 antibodies. Anti-IL-6 receptor antibody significantly (but only partially) suppressed T cell activation (as indicated by [3H]-thymidine uptake and CD25 expression) and IL-2 production in both systems, and increased the frequency of regulatory T cells among spleen cells. Anti-TNFalpha antibody had no effects in either system. Neither antibody increased the expression of markers of apoptosis in CD4 T cells. In conclusion, our results show that anti-IL-6 receptor antibody significantly (but only partially) suppressed the T cell receptor signalling-induced activation of CD4 T cells and also suggest that it achieved this partial suppression by the partial inhibition of IL-2 production and the induction of regulatory T cells. In stark contrast, anti-TNFalpha antibody had no impact on T cell activation. Extrapolating these results to the clinical treatment of rheumatoid arthritis, they suggest that IL-6 blockade inhibits T cell activation, whereas TNFalpha blockade does not.

Vitamin D Receptor Signaling Enhances Locomotive Ability in Mice

Bone fractures markedly reduce quality of life and life expectancy in elderly people. Although osteoporosis increases bone fragility, fractures frequently occur in patients with normal bone mineral density. Because most fractures occur on falling, preventing falls is another focus for reducing bone fractures. In this study, we investigated the role of vitamin D receptor (VDR) signaling in locomotive ability. In the rotarod test, physical exercise enhanced locomotive ability of wild‐type (WT) mice by 1.6‐fold, whereas exercise did not enhance locomotive ability of VDR knockout (KO) mice. Compared with WT mice, VDR KO mice had smaller peripheral nerve axonal diameter and disordered AChR morphology on the extensor digitorum longus muscle. Eldecalcitol (ED‐71, ELD), an analog of 1,25(OH)2D3, administered to rotarod‐trained C57BL/6 mice enhanced locomotor performance compared with vehicle‐treated nontrained mice. The area of AChR cluster on the extensor digitorum longus was greater in ELD‐treated mice than in vehicle‐treated mice. ELD and 1,25(OH)2D3 enhanced expression of IGF‐1, myelin basic protein, and VDR in rat primary Schwann cells. VDR signaling regulates neuromuscular maintenance and enhances locomotive ability after physical exercise. Further investigation is required, but Schwann cells and the neuromuscular junction are targets of vitamin D3 signaling in locomotive ability.

Intercellular adhesion molecule‐1 on synovial cells attenuated interleukin‐6‐induced inhibition of osteoclastogenesis induced by receptor activator for nuclear factor κB ligand

In a co‐culture of osteoclast precursor cells and synovial cells, interleukin‐6 (IL‐6) induces osteoclast formation. In contrast, in a monoculture of osteoclast precursor cells, IL‐6 directly suppresses receptor activator for nuclear factor κB ligand (RANKL)‐induced differentiation of osteoclast precursor cells into osteoclasts. In the present study, we explored why the effect of IL‐6 differed between the monoculture and the co‐culture systems. In the monoculture, mouse osteoclast precursor cell line, RAW 264·7 (RAW) cells were cultured with soluble RANKL (sRANKL) for 24 h or 3 days. sRANKL increased both expression of osteoclastogenesis marker, tartrate‐resistant acid phosphatase isoform 5b (TRAP5b) and nuclear factor of activated T cells cytoplasmic 1 (NFATc1), whereas the co‐addition of IL‐6 decreased them both in a dose‐dependent manner. In the co‐culture, RAW cells and human synovial cell line, SW982 cells were cultured with IL‐6 + soluble IL‐6 receptor (sIL‐6R) for 3 days. TRAP5b and NFATc1 expression reduced by IL‐6 was increased by the addition of SW982 cells in a manner dependent upon the number of added cells. IL‐6 + sIL‐6R treatment significantly induced RANKL production in SW982 cells, and anti‐RANKL antibody inhibited IL‐6 + sIL‐6R‐induced osteoclastogenesis. SW982 cells expressed high levels of ICAM‐1 originally, and ICAM‐1 expression was increased significantly by IL‐6 + sIL‐6R. Anti‐ICAM‐1 antibody suppressed IL‐6‐induced osteoclastogenesis. Finally, in the monoculture system, addition of sICAM‐1 dose‐dependently restored the expression of TRAP5b reduced by IL‐6. Similar results were obtained when the formation of TRAP‐positive multi‐nuclear cells were examined using mouse bone marrow cells. In conclusion, IL‐6 gave different results in the co‐culture and monoculture systems because in the co‐culture, ICAM‐1 from the synovial cells restored osteoclastogenesis suppressed by IL‐6.

Adiponectin induces CCL20 expression synergistically with IL-6 and TNF-α in THP-1 macrophages.

Adiponectin (Ad) is an adipokine secreted from adipocytes. It is reported that Ad has many biological activities. However, its influence on inflammation is controversial. In the present study, we examined the influence of Ad on production of CCL20 from THP-1 macrophages. THP-1 macrophages were prepared from THP-1 monocytes by PMA treatment. THP-1 macrophages were cultured for 24h with Ad, IL-6, or TNF-α alone or with combinations of Ad and cytokines. CCL20 mRNA expression was then determined by real-time PCR. Full-length Ad (fAd) slightly but significantly induced CCL20 mRNA expression, and interestingly, co-stimulation with fAd and IL-6 or with fAd and TNF-α synergistically increased the expression of CCL20 mRNA. We explored the mechanism behind the synergistic effect of fAd and these cytokines. fAd did not affect the expression of receptors for IL-6 and TNF, and IL-6 and TNF-α did not increase the expression of the receptor for Ad in THP-1 macrophages. The increased expression of CCL20 by fAd is much higher in THP-1 macrophages compared with THP-1 monocytes. Furthermore, MMP-12 production was increased by IL-6 and TNF-α in THP-1 macrophages but it was not detectable in THP-1 monocytes. Treatment of fAd with MMP-12 induced globular Ad (gAd), and the expression of CCL20 in THP-1 macrophages was increased more potently by gAd than by fAd. MMP inhibitor (UK370106) inhibited the expression of CCL20 induced by co-stimulation with fAd and IL-6 or TNF-α. In conclusion, gAd played an important role in CCL20 expression, and MMP-12 induced by IL-6 or TNF-α was involved in the synergistic effect of fAd and cytokines.

Interleukin-6 regulates anti-arthritic effect of methotrexate via reduction of SLC19A1 expression in a mouse arthritis model

IntroductionMethotrexate (MTX) enters cells via the reduced folate carrier SLC19A1, suggesting that SLC19A1 is associated with the efficacy of MTX. We here examined the relationship between the efficacy of MTX and the expression of SLC19A1 in glucose 6-phosphate isomerase (GPI)-induced arthritis. We found that interleukin-6 (IL-6) regulated the expression of SLC19A1, so we studied the effect of a combination of MTX and anti-mouse IL-6 receptor antibody (MR16-1).MethodsGPI-induced arthritis was induced by intradermal immunization with recombinant GPI. MTX was given from the first day of immunization. Mice were injected once with MR16-1 10 days after immunization. The levels of SLC19A1 mRNA in whole hind limbs and immune cells were measured. Synovial cells from arthritic mice were cultured with cytokines, and cell proliferation and gene expressions were measured.ResultsMTX inhibited the development of GPI-induced arthritis; however, the efficacy of MTX gradually diminished. SLC19A1 expression in immunized mice with arthritis was lower than in intact mice; moreover, SLC19A1 expression in arthritic mice was further decreased when they were treated with MTX. IL-6 was highly expressed in whole hind limbs of arthritic mice. In an in vitro study using synovial cells from arthritic mice, IL-6 + soluble IL-6 receptor (sIL-6R) weakened the anti-proliferative effect of MTX and reduced SLC19A1 expression. Finally, although MR16-1 did not improve arthritis at all when administered on day 10, MTX in combination with MR16-1 more potently reduced the development of arthritis than did MTX alone. When used in combination with MTX, MR16-1 apparently reversed the decrease in SLC19A1 induced by MTX alone.ConclusionsIn the present study, we demonstrated for the first time that IL-6 reduced the efficacy of MTX by decreasing the expression of SLC19A1, which is important for MTX uptake into cells.

THU0061 The Effect of Anti-IL-6 Receptor Antibody on Cartilage Destruction in a Mouse Model of Collagen-Induced Arthritis

Background Reports of clinical trials for RA show that the anti-IL-6 receptor antibody tocilizumab (anti-IL-6R) inhibits not only bone erosion but also joint space narrowing [1]. Cartilage destruction is thought to be partly caused by increased expression of enzymes that degrade the cartilage matrix. It has also been reported that oxidative stress is increased in patients with RA [2] and plays a key role in cartilage destruction [3]. However, it is not yet clear how anti-IL-6R inhibits cartilage destruction. Objectives The purpose of this study was to investigate the effect of anti-IL-6R on cartilage destruction and the levels of the cartilage matrix–degrading enzymes and oxidative stress in a mouse model of collagen-induced arthritis (CIA). Methods After allocating some mice to a normal group, CIA was triggered in DBA/1J mice by an intradermal injection of bovine type II collagen, and these mice were allocated to a control group or a treated group. Mice in the treated group were injected intraperitoneally with anti-mouse IL-6 receptor antibody (MR16-1). Samples of serum and hind limbs were taken at the peak of swelling (Day 36). The levels of cartilage matrix–degrading enzymes (MMP-3, MMP-13, ADAMTS4, and ADAMTS5) in serum were measured by ELISA. The level of oxidative stress – as indicated by derivatives of reactive oxygen metabolites (d-ROMs) in serum – was measured using a free radical analytical system. The length of the joint space in the hind limbs was analysed by micro-computed tomography. Results In the control group, MMP-3, MMP-13, ADAMTS5, and d-ROM were significantly higher than in the normal group. ADAMTS4 could not be detected in any mouse. The space between joints in the hind limbs was significantly narrower in the control group than in the normal group, and thicker in the MR16-1 group than in the control group (Figure 1 arrows). The arthritis score and the levels of MMP-3, MMP-13, ADAMTS5, and d-ROM were lower in the MR16-1 group than in the control group. Conclusions We demonstrated that CIA-induced joint space narrowing was accompanied by increased expression of cartilage matrix–degrading enzymes and oxidative stress. Furthermore, our results also indicated that IL-6 plays an important role in cartilage destruction. Our findings indicate that anti-IL-6R may suppress cartilage destruction by inhibiting the increased expression of cartilage matrix–degrading enzymes and oxidative stress. References Kremer et al. Arthritis Rheum. 2011; 63: 609-621. Hitchon CA et al. Arthritis Res Ther. 2004; 6: 265-278. Reed KN et al. Mol Cell Biochem. 2014; 397: 195-201. Disclosure of Interest None declared