Hiroto Yoshida

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 blockade inhibits the induction of myelin antigen-specific Th17 cells and Th1 cells in experimental autoimmune encephalomyelitis

The development of Th17 cells is a key event in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a murine model of human multiple sclerosis (MS). Previous studies have demonstrated that an IL-6-dependent pathway is involved in the differentiation of Th17 cells from naïve CD4-positive T cells in vitro. However, the role of IL-6 in vivo in the development of Th17 cells in EAE has remained unclear. In the present study, we found that IL-6 blockade by treatment with an anti-IL-6 receptor monoclonal antibody (anti-IL-6R mAb) inhibited the development of EAE and inhibited the induction of myelin oligodendrocyte glycoprotein (MOG) peptide-specific CD4-positive, CD8-positive, and Th17 T cells, in inguinal lymph nodes. Thus, the protective effect of IL-6 blockade in EAE is likely to be mediated via the inhibition of the development of MOG-peptide-specific Th17 cells and Th1 cells, which in turn leads to reduced infiltration of T cells into the CNS. These findings indicate that anti-IL-6R mAb treatment might represent a novel therapy for human MS.

Interleukin-6 blockade suppresses autoimmune arthritis in mice by the inhibition of inflammatory Th17 responses

OBJECTIVE To investigate the mechanism of interleukin-6 (IL-6) blockade in autoimmune arthritis, by comparing the effect of anti-IL-6 receptor (anti-IL-6R) monoclonal antibody (mAb) treatment with the effect of soluble tumor necrosis factor (sTNFR)-Fc fusion protein treatment on T helper cell differentiation in collagen-induced arthritis (CIA). METHODS DBA/1 mice were immunized with type II collagen (CII) to induce arthritis and were left untreated or were treated with anti-IL-6R mAb or TNFR-Fc. T helper cell differentiation and cytokine expression during the development of arthritis in these mice were analyzed. RESULTS Immunization with CII predominantly increased the frequency of Th17 cells rather than Th1 cells. The frequency of FoxP3+ Treg cells was also increased after immunization. Treatment of mice with CIA with anti-IL-6R mAb on day 0 markedly suppressed the induction of Th17 cells and arthritis development, but treatment with this antibody on day 14 failed to suppress both Th17 differentiation and arthritis. In contrast, treatment of mice with CIA with TNFR-Fc from day 0 to day 14 suppressed neither Th17 differentiation nor arthritis, but treatment from day 21 to day 35 successfully ameliorated arthritis without inhibiting Th17 induction. Neither antibody treatment increased the frequency of Treg cells. CONCLUSION Our results indicate that the protective effect of IL-6 blockade, but not tumor necrosis factor (TNF) blockade, in CIA correlates with the inhibition of Th17 differentiation. Our findings suggest that IL-6 blockade in rheumatoid arthritis in human is also likely to involve a therapeutic mechanism distinct from that of TNF blockade and thus may represent an alternative therapy for patients in whom the disease is refractory to TNF blockade.

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-IL-6 receptor antibody increases blood IL-6 level via the blockade of IL-6 clearance, but not via the induction of IL-6 production

We explored the mechanism for the increase of blood IL-6 level after anti-IL-6 receptor (IL-6R) antibody injection. First, we examined whether anti-IL-6R antibody stimulates IL-6 production. Single injection of tocilizumab (anti-IL-6R antibody) in monkeys with collagen-induced arthritis (CIA) caused a marked increase in blood IL-6 and IL-6R levels, but did not increase IL-6 mRNA and IL-6R mRNA expression in liver, spleen, lymph nodes, synovium or whole blood 1, 3 and 7 days later. This suggests that tocilizumab did not induce IL-6 and IL-6R production. Second, we investigated whether anti-IL-6R antibody releases IL-6 from IL-6 complexes in the blood. When plasma from CIA monkeys was incubated with tocilizumab, the IL-6 concentration was not affected. Finally, we studied whether anti-IL-6R antibody affects the clearance of IL-6 from the blood. When MR16-1 (anti-mouse IL-6R antibody) was injected into IL-6-deficient mice continuously infused with human IL-6, blood human IL-6 levels significantly increased. These results suggest that the elevation of blood IL-6 after the administration of anti-IL-6R antibody is the result of inhibition of the clearance of IL-6 due to IL-6R blockade, and that it is not the result of induction of IL-6 production or release of IL-6 from complexes.

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.

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.

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.