Masashi Shiina

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 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.

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.

FRI0048 Interleukin-6 increases the number of osteoclast precursors in femur bone marrow via up-regulation of sphingosine-1-phosphate receptor 2 in inflammatory arthritic mice

Background Rheumatoid arthritis (RA) is associated with an increased risk of osteoporosis and fractures. A number of factors such as menopause, glucocorticoid use, and high RA disease activity are associated with an increased risk of osteoporosis. Several studies have demonstrated a link between osteoporosis and pro-inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Recent studies have shown that sphingosine-1-phosphate (S1P) controls the dynamic intermigration of osteoclast precursors (OCPs) between the blood and bones, in part via the S1P receptor 2 (S1PR2) expressed on the surface of OCPs. Inhibition of S1PR2 function changed the dynamics of OCP migration and relieved osteoporosis in a mouse model of bone loss. However, it is not yet understood whether pro-inflammatory cytokines affect the expression and function of S1PR2 in the course of bone loss. Objectives The purpose of this study was to investigate whether IL-6 and TNF-α regulate the expression and function of S1PR2, and whether they have any influence on the localization of OCPs in the course of bone loss. Methods OCPs (CD11b+Gr-1low+med) were isolated from DBA/1J mice bone marrow by using a cell sorter, and were stimulated with IL-6 or TNF-α. S1PR2 mRNA expression in OCPs was measured by real-time PCR. To examine the function of S1PR2, S1P-directed chemotactic activity was evaluated by using a Transwell plate. DBA/1J mice were immunized intradermally with bovine type II collagen (Day 0), and anti-mouse IL-6 receptor antibody and control IgG were administered intraperitoneally on the same day. On Days 8 and 15, femurs were excised and the trabecular bone volume (bone volume/tissue volume) of the distal femur was analyzed using microfocus computed tomography. The number of OCPs in the femur bone marrow was counted by flow cytometry. S1PR2 expression in OCPs from immunized mice was measured by real-time PCR. Results IL-6 induced S1PR2 in OCPs in a dose-dependent manner, but TNF-α did not induce S1PR2. Because S1PR2 negatively regulates chemotaxis in the direction of S1P, which is rich in blood, we examined the migratory response of OCPs stimulated by IL-6 in the direction of S1P. In the presence of IL-6, S1P-directed chemotaxis of OCPs in the direction of S1P was decreased by 64% compared with medium control. Trabecular bone volume in immunized mice was decreased by more than 9% on Day 8 and was significantly decreased by 33% on Day 15 compared with normal mice. The number of OCPs in femur bone marrow significantly increased in immunized mice at Day 8 and Day 15; moreover, the expression of S1PR2 in immunized mice was significantly increased compared with normal mice. Treatment of immunized mice with anti-IL-6 receptor antibody decreased the number of OCPs in femur bone marrow by 25% and the expression of S1PR2 by 27%; however no changes were seen following treatment with control IgG. Conclusions The results demonstrated that IL-6 increases the number of OCPs in femur bone marrow via up-regulating S1PR2, and plays a crucial role in bone loss induced by inflammation. Disclosure of Interest None Declared

THU0079 Anti-IL-6 Receptor Antibody Suppresses Systemic Bone Loss by not Only Normalizing Bone Resorption but also Enhancing Bone Formation in a Mouse Model of Collagen-Induced Arthritis

Background Patients with rheumatoid arthritis (RA) have a high risk of osteoporosis and osteoporotic fracture. In primary osteoporosis (either high bone turnover osteoporosis [type I] or low bone turnover osteoporosis [type II]), bone loss is induced by an imbalance of bone metabolism: bone resorption dominates bone formation. However, it is not fully understood how RA affects bone metabolism or if bone metabolism is improved by blockade of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Objectives We aimed to examine change in the balance between bone resorption markers and bone formation markers in inflammation, and how it is affected by blockade of IL-6 or TNF-α, using a mouse model of collagen-induced arthritis (CIA). Methods CIA in DBA/1J mice was triggered by intradermal injection of bovine type II collagen on day 0 and 21. Mice were injected with anti-mouse IL-6 receptor antibody (MR16-1) intraperitoneally on days 0 and 21. On the other hand, TNF receptor-Fc (TNFR-Fc) was given intraperitoneally 3 times per week from the first immunization. Serum and urine were sampled on days 14 (before swelling), 35 (peak of swelling), and 54 (after swelling subsides). Urinary bone resorption markers (DPD: deoxypyridinoline, CTX: collagen type 1 cross-linked C-telopeptide) and serum bone formation markers (P1NP: procollagen type 1 N-terminal propeptide, OC: osteocalcin) were measured by ELISA. The lumbar spine was excised on day 56, and trabecular bone volume (BV/TV) was analysed by micro-computed tomography (μCT). Results In CIA mice, lumbar spine BV/TV on day 56 had significantly declined by 62.1% compared with non-immunized mice. Both MR16-1 and TNFR-Fc significantly suppressed development of arthritis compared with untreated CIA mice. In MR16-1-treated mice, lumbar spine BV/TV was significantly increased to 1.44 times that of untreated CIA mice; TNFR-Fc also increased BV/TV (1.13 times), but not significantly. On days 35 and 54, CTX and DPD levels were significantly higher in CIA mice than in non-immunized mice. OC level in CIA mice was significantly lowered on day 14. On day 35, OC had recovered to the level in non-immunized mice; however, P1NP was significantly lower. In MR16-1-treated mice, bone resorption markers were significantly lower on days 35 (CTX) and 54 (CTX and DPD) than in untreated CIA mice, and bone formation markers were significantly higher on days 14 (P1NP) and 35 (P1NP and OC). Moreover, bone formation markers were higher on day 35 than in non-immunized mice. In TNFR-Fc-treated mice, on the other hand, although bone resorption markers on days 35 (CTX) and 54 (DPD) were significantly decreased, bone formation marker levels were similar to those of untreated CIA mice. Conclusions We demonstrated that CIA induced severe bone loss by an imbalance of bone metabolism, not only increasing bone resorption but also suppressing bone formation. Moreover, our results indicated that both IL-6 and TNF play an important role in increasing bone resorption, but that only IL-6 played a crucial role in decreasing bone formation. Our findings provide evidence that anti-IL-6 receptor antibody would have a beneficial effect on systemic osteoporosis in RA patients. Disclosure of Interest None Declared