Misato Hashizume

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.

IL-6 trans-signalling directly induces RANKL on fibroblast-like synovial cells and is involved in RANKL induction by TNF-α and IL-17

OBJECTIVES We investigated the influence of cytokines on the expression of RANK ligand (RANKL) in fibroblast-like synoviocytes from RA patients (RA-FLS). METHODS RA-FLS were stimulated by IL-6, TNF-alpha, IL-17 and IL-1beta with or without soluble IL-6 receptor (sIL-6R) for 24 h. The expression of RANKL was measured by real-time PCR, western blotting and immunostaining. In proliferation assay, RA-FLS were cultured with cytokines for 3 days. RA-FLS were co-cultured with RAW cell in the presence of IL-6/sIL-6R for 3 days and then NFATc1 mRNA expression in RAW cells was examined. RA-FLS was cultured with parthenolide [PAR, signal transducer and activator of transcription (STAT) inhibitor] or PD98059 (PD, mitogen-activated protein kinase inhibitor) in the presence of IL-6/sIL-6R and then the influence of these drugs on phosphorylation of STAT3 and ERK1/2, and RANKL expression was examined. RESULTS RANKL expression was induced by IL-6/sIL-6R (but not IL-6 alone) and by IL-1beta. On the other hand, TNF-alpha and IL-17 did not induce RANKL expression, although TNF-alpha, IL-17 or IL-1beta stimulated cell growth and IL-6 production. However, in the presence of sIL-6R, TNF-alpha or IL-17 induced RANKL expression. By the co-culture of RA-FLS, NFATc1 mRNA expression was induced in RAW cells. Finally, IL-6/sIL-6R induced phosphorylation of STAT3 and ERK1/2 in RA-FLS, and was completely inhibited by PAR and PD, respectively. PAR completely inhibited IL-6/sIL-6R-induced RANKL expression, but PD did not. CONCLUSIONS IL-6/sIL-6R directly induced RANKL expression in RA-FLS and it is essential for RANKL induction by TNF-alpha and IL-17. Moreover, RANKL induction by IL-6/sIL-6R is mediated by the janus kinase/STAT signalling pathway.

The Roles of Interleukin-6 in the Pathogenesis of Rheumatoid Arthritis

Several clinical studies have demonstrated that the humanized anti-interleukin-6 (IL-6) receptor antibody tocilizumab (TCZ) improves clinical symptoms and prevents progression of joint destruction in rheumatoid arthritis (RA). However, the precise mechanism by which IL-6 blockade leads to the improvement of RA is not well understood. IL-6 promotes synovitis by inducing neovascularization, infiltration of inflammatory cells, and synovial hyperplasia. IL-6 causes bone resorption by inducing osteoclast formation via the induction of RANKL in synovial cells, and cartilage degeneration by producing matrix metalloproteinases (MMPs) in synovial cells and chondrocytes. Moreover, IL-6 is involved in autoimmunity by altering the balance between Th17 cells and Treg. IL-6 also acts on changing lipid concentrations in blood and on inducing the production of hepcidin which causes iron-deficient anemia. In conclusion, IL-6 is a major player in the pathogenesis of RA, and current evidence indicates that the blockade of IL-6 is a beneficial therapy for RA patients.

High molecular weight hyaluronic acid inhibits IL-6-induced MMP production from human chondrocytes by up-regulating the ERK inhibitor, MKP-1

To investigate the mechanism of the inhibitory action of high molecular weight hyaluronic acid (HA) on production of matrix metalloproteinases (MMPs) induced by IL-6 in human chondrocyte. Human chondrocyte were stimulated by interleukin-6 (IL-6) and soluble IL-6 receptor (sIL-6R) with or without HA for 24h and the productions of MMP-1, MMP-3 and MMP-13 were measured. Phosphorylations of extracellular signal-regulated kinase (ERK), signal transducer and activator of transcription (STAT) and mitogen-activated protein kinase kinase (MEK) in IL-6+sIL-6R-treated chondrocytes were detected by western blotting. IL-6+sIL-6R induced MMP-1, MMP-3 and MMP-13 productions from human chondrocyte. Inhibition of the mitogen-activated protein kinase (MAPK) signaling pathway resulted in marked decreases of MMP-1, MMP-3 and MMP-13 induction by IL-6. In contrast, STAT inhibition only slightly attenuated the production of MMPs. HA inhibited MMP-1, MMP-3 and MMP-13 induction by IL-6, which was reversed by the addition of anti-CD44 antibody but not anti-ICAM-1 antibody. Pre-treatment of cells with HA reduced the phosphorylation of ERK, but not MEK. Expression levels of mitogen-activated protein kinase phosphatase-1 (MKP-1) in HA-treated chondrocytes were assessed by western blotting. HA induced the expression of MKP-1, a negative regulator of ERK1/2 in IL-6+sIL-6R-treated or untreated chondrocytes, and the MKP-1 inhibitor and MKP-1 siRNA reversed the HA-induced suppression of MMP induction by IL-6. Our study is the first to demonstrate that HA suppressed MMPs induction by IL-6 in human chondrocyte via MKP-1 induction through CD44 signaling.

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.

Atherogenic effects of TNF-α and IL-6 via up-regulation of scavenger receptors.

Patients with chronic inflammatory disorders such as rheumatoid arthritis (RA) have a high risk of developing cardiovascular disease. We evaluated the effects of TNF-α and IL-6 on foam cell formation, a pivotal process in atherogenesis. Accumulation of intracellular oxidized LDL (oxLDL) was induced when THP-1/macrophages were stimulated with TNF-α or IL-6. TNF-α induced the expressions of scavenger receptors SR-A and LOX-1, and IL-6 induced SR-A expression. Inhibition of the NF-κB signaling markedly decreased TNF-α-induced foam cell formation and SR-A expression. Serum from RA patients, but not healthy subjects, induced foam cell formation, which was partially reversed by either IL-6 or TNF-α blockade in conjunction with inhibiting the induction of scavenger receptors. The present study clearly showed that in patients with chronic inflammation mediated by TNF-α and IL-6, these cytokines are directly implicated in atherosclerotic plaque formation.

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.

IL-6 plays an essential role in neutrophilia under inflammation.

In the present study, we explored the involvement of interleukin-6 (IL-6) in neutrophilia under inflammatory conditions. The neutrophil count in the peripheral blood was high in arthritic monkeys, and anti-IL-6 receptor antibody reduced neutrophil counts to normal levels. IL-6 injection into normal monkeys significantly increased neutrophil counts in the blood 3h after injection. The expression of cluster of differentiation (CD) 162 on circulating neutrophils was reduced by IL-6 injection. IL-6 treatment in vitro did not affect CD162 expression on neutrophils from human blood. In IL-6-treated monkeys, IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels in plasma were clearly elevated. IL-8 and GM-CSF treatment in vitro reduced cell-surface CD162 expression on human neutrophils, and moreover, increased soluble CD162 expression in the cell supernatant. The addition of IL-6 into human whole peripheral blood induced IL-8 production and reduced CD162 expression on neutrophils. Furthermore, IL-8 and GM-CSF augmented mRNA expression of a disintegrin and metalloprotease like domain 10 (ADAM10) in neutrophils. Knock-down of ADAM10 by siRNA in neutrophil-like HL-60 cells partially reversed the expression of CD162 reduced by GM-CSF and IL-8 on HL-60 cells. In conclusion, IL-6 induced neutrophilia and reduced CD162 expression on neutrophils in inflammation.

Baseline levels of soluble interleukin-6 receptor predict clinical remission in patients with rheumatoid arthritis treated with tocilizumab: implications for molecular targeted therapy

Interleukin-6 (IL-6) is a monomeric protein that binds to either soluble or membrane-bound IL-6 receptors (IL-6R)1 ,2 Tocilizumab, a humanised anti-IL-6R monoclonal antibody, binds to soluble IL-6R (sIL-6R) and membrane-bound IL-6R, blocking signal transduction pathways through competitive inhibition of IL-6 binding.3 We have previously demonstrated that baseline plasma tumour necrosis factor (TNF) levels are associated with the clinical response to infliximab, anti-TNF monoclonal antibody binding to soluble and membrane-bound TNF.4 Therefore, it is tempting to speculate that baseline serum levels of sIL-6R, rather than those of IL-6, are associated with clinical response to tocilizumab in patients with rheumatoid arthritis (RA). To test this hypothesis, we analysed serum levels of IL-6 and sIL-6R before tocilizumab treatment in our institution and evaluated their association with clinical remission. Consecutive patients with RA in our institution who commenced 8 mg/kg tocilizumab treatment every 4 weeks as the first biologic agent between March 2010 and April 2012 were included. At baseline, serum levels of IL-6 and sIL-6R were measured by electrochemiluminescence assay with the Ultra-Sensitive Kit (Meso Scale …

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.