Yoshiyuki Ohsugi

Blockage of interleukin-6 receptor ameliorates joint disease in murine collagen-induced arthritis

OBJECTIVE To clarify the role of interleukin-6 (IL-6) in the pathogenesis of collagen-induced arthritis (CIA). METHODS CIA was induced by immunizing twice at a 3-week interval with bovine type II collagen (CII) emulsified with complete adjuvant. Rat anti-mouse IL-6 receptor (anti-IL-6R) monoclonal antibody MR16-1 or isotype-matched control antibody KH-5 was then injected once intraperitoneally. Symptoms of arthritis were evaluated with a visual scoring system, and serum anti-CII antibody and IL-6 levels were measured by enzyme-linked immunosorbent assay. In addition, the CII responsiveness of splenic lymphocytes from mice with CIA was examined. RESULTS In mice with CIA, excess production of IL-6 in sera was observed within 24 hours after the first CII immunization, and then rapidly decreased. Serum IL-6 increased again beginning 14 days after immunization, in conjunction with the onset of arthritis. When MR16-1 was injected immediately after immunization with CII, it inhibited the development of arthritis in a dose-dependent manner. Furthermore, MR16-1-treated mice exhibited lower serum levels of IgG anti-CII antibody and reduced responsiveness of lymphocytes to CII. This suppressive effect was observed when MR16-1 was injected on day 0 or 3, but not when injected on day 7 or 14. CONCLUSION IL-6 produced after CII immunization appears to play an essential role in the immunity to CII, and anti-IL-6R antibody reduces the development of CIA by suppressing IL-6 signal transduction.

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.

Blockade of interleukin-6 receptor suppresses reactive astrogliosis and ameliorates functional recovery in experimental spinal cord injury.

Endogenous neural stem/progenitor cells (NSPCs) have recently been shown to differentiate exclusively into astrocytes, the cells that are involved in glial scar formation after spinal cord injury (SCI). The microenvironment of the spinal cord, especially the inflammatory cytokines that dramatically increase in the acute phase at the injury site, is considered to be an important cause of inhibitory mechanism of neuronal differentiation following SCI. Interleukin‐6 (IL‐6), which has been demonstrated to induce NSPCs to undergo astrocytic differentiation selectively through the JAK/STAT pathway in vitro, has also been demonstrated to play a critical role as a proinflammatory cytokine and to be associated with secondary tissue damage in SCI. In this study, we assessed the efficacy of rat anti‐mouse IL‐6 receptor monoclonal antibody (MR16‐1) in the treatment of acute SCI in mice. Immediately after contusive SCI with a modified NYU impactor, mice were intraperitoneally injected with a single dose of MR16‐1 (100 μg/g body weight), the lesions were assessed histologically, and the functional recovery was evaluated. MR16‐1 not only suppressed the astrocytic diffentiation‐promoting effect of IL‐6 signaling in vitro but inhibited the development of astrogliosis after SCI in vivo. MR16‐1 also decreased the number of invading inflammatory cells and the severity of connective tissue scar formation. In addition, we observed significant functional recovery in the mice treated with MR16‐1 compared with control mice. These findings suggest that neutralization of IL‐6 signaling in the acute phase of SCI represents an attractive option for the treatment of SCI.

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.

Blockade of interleukin-6 signaling aggravates ischemic cerebral damage in mice: possible involvement of Stat3 activation in the protection of neurons

Interleukin (IL)‐6 expression transiently increases in the acute phase of cerebral ischemia. To investigate the physiological significance of endogenous IL‐6 expression and to identify the main signal pathway for the action of IL‐6, we administered anti‐mouse IL‐6 receptor monoclonal antibody (IL‐6RA), which blocks IL‐6 signaling, to mice immediately after a 45‐min period of middle cerebral artery occlusion (MCAO). At 6 h after MCAO, IL‐6RA administration had resulted in a significant reduction in the amount of phosphorylated signal transducer and activator of transcription‐3 (Stat3) protein in the peri‐infarct area of the cortex. At 24 h after MCAO, blockade of IL‐6 signaling had led to an increase in number of apoptotic cells in the peri‐infarct area and enlargement of the size of the infarct, and it had adversely affected neurological function. These results suggest that endogenous IL‐6 plays a critical role in preventing damaged neurons from undergoing apoptosis in the acute phase of cerebral ischemia and that its role may be mediated by Stat3 activation.

IL‐6 receptor blockage inhibits the onset of autoimmune kidney disease in NZB/W F1 mice

In the present study, we examined the preventive effect of anti‐mouse IL‐6 receptor (IL‐6R) antibody, MR16‐1, on the development of autoimmune kidney disease in female NZB/W F1 (BWF1) mice. Immunological tolerance to MR16‐1 or isotype‐matched control antibody, KH‐5, was induced by the simultaneous administration of anti‐CD4 MoAb in mice. Thereafter, mice were intraperitoneally given 0.5 mg of MR16‐1, 0.5 mg of KH‐5 or saline once a week from 13 to 64 weeks of age. MR16‐1 treatment dramatically suppressed proteinuria and prolonged the survival time of BWF1 mice. Only one out of 10 mice died with high levels of proteinuria throughout the experiment. MR16‐1 almost completely suppressed the production of IgG forms of anti‐DNA and anti‐TNP antibodies, but not the IgM forms of these antibodies. In particular, all IgG subclasses (IgG1, IgG2a, IgG2b and IgG3) of anti‐DNA antibody production were significantly suppressed. Moreover, serum IgG1, IgG2a and IgG3 levels in MR16‐1‐treated mice were lower than those in saline‐ and KH‐5‐treated mice, whereas serum IgM and IgA levels were not influenced. In conclusion, MR16‐1 potently suppressed the development of autoimmune disease in BWF1 mice, and this was attributed to its effect of specific suppression of IgG class antibody production.

ADP ribosyl cyclase activity of a novel bone marrow stromal cell surface molecule, BST-1

Human BST‐1, a bone marrow stromal cell surface molecule, is a GPI‐anchored protein that facilitates the growth of pre‐B cells. The deduced amino acid sequences of human and mouse BST‐1 show around 30% homology with those of CD38 and Aplysia ADP ribosyl cyclase. Therefore, like CD38, BST‐1 might possess ADP ribosyl cyclase activity. Here, we report the establishment of a stable transformant CHO cell line which secretes truncated human soluble BST‐1, and show that purified soluble BST‐1 displays both ADP ribosyl cyclase cADPR hydrolase activities.

Crucial role of the interleukin-6/interleukin-17 cytokine axis in the induction of arthritis by glucose-6-phosphate isomerase

OBJECTIVE To clarify the glucose-6-phosphate isomerase (GPI)-specific CD4+ T cell lineage involved in GPI-induced arthritis and to investigate their pathologic and regulatory roles in the induction of the disease. METHODS DBA/1 mice were immunized with GPI to induce arthritis. CD4+ T cells and antigen-presenting cells were cocultured with GPI, and cytokines in the supernatant were analyzed by enzyme-linked immunosorbent assay. Anti-interferon-gamma (anti-IFNgamma) monoclonal antibody (mAb), anti-interleukin-17 (anti-IL-17) mAb, or the murine IL-6 receptor (IL-6R) mAb MR16-1 was injected at different time points, and arthritis development was monitored visually. After MR16-1 was injected, percentages of Th1, Th2, Th17, and Treg cells were analyzed by flow cytometry, and CD4+ T cell proliferation was analyzed using carboxyfluorescein diacetate succinimidyl ester. RESULTS GPI-specific CD4+ T cells were found to be differentiated to Th1 and Th17 cells, but not Th2 cells. Administration of anti-IL-17 mAb on day 7 significantly ameliorated arthritis (P < 0.01), whereas administration of anti-IFNgamma mAb exacerbated arthritis. Neither anti-IL-17 mAb nor anti-IFNgamma mAb administration on day 14 ameliorated arthritis. Administration of MR16-1 on day 0 or day 3 protected against arthritis induction, and MR16-1 administration on day 8 significantly ameliorated existing arthritis (P < 0.05). After administration of MR16-1, there was marked suppression of Th17 differentiation, without an increase in Th1, Th2, or Treg cells, and CD4+ T cell proliferation was also suppressed. CONCLUSION IL-6 and Th17 play an essential role in GPI-induced arthritis. Since it has previously been shown that treatment with a humanized anti-IL-6R mAb has excellent effects in patients with rheumatoid arthritis (RA), we propose that the IL-6/IL-17 axis might also be involved in the generation of RA, especially in the early effector phase.

Anti-interleukin-6 receptor antibody prevents muscle atrophy in colon-26 adenocarcinoma-bearing mice with modulation of lysosomal and ATP-ubiquitin-dependent proteolytic pathways

Progression of skeletal muscle atrophy is one of the characteristic features in cancer patients. Interleukin‐6 (IL‐6) has been reported to be responsible for the loss of lean body mass during cancer cachexia in colon‐26 adenocarcinoma (C‐26)‐bearing mice. This study was carried out to elucidate the intracellular proteolytic pathways operating in skeletal muscle in C‐26‐bearing mice, and to examine the effect of anti IL‐6 receptor antibody on muscle atrophy. On day 17 after tumor inoculation, the gastrocnemius muscle weight of C‐26‐bearing mice had significantly decreased to 69% of that of the pair‐fed control mice. This weight loss occurred in association with increases in the mRNA levels of cathepsins B and L, poly‐ubiquitin (Ub) and the subunits of proteasomes in the muscles. Furthermore, enzymatic activity of cathepsin B+L in the muscles also increased to 119% of the control. The administration of antimurine IL‐6 receptor antibody to C‐26‐bearing mice reduced the weight loss of the gastrocnemius muscles to 84% of that of the control mice, whose enzymatic activity of cathepsin B+L and mRNA levels of cathepsin L and poly‐Ub were significantly suppressed compared with those of the C‐26‐bearing mice. Our data indicate that both the lysosomal cathepsin pathway and the ATP‐dependent proteolytic pathway might be involved in the muscle atrophy of C‐26‐bearing mice. The results also suggest that anti IL‐6 receptor antibody could be a potential therapeutic agent against muscle atrophy in cancer cachexia by inhibiting these proteolytic systems.

Anti-IL-6-receptor antibody promotes repair of spinal cord injury by inducing microglia-dominant inflammation

We previously reported the beneficial effect of administering an anti-mouse IL-6 receptor antibody (MR16-1) immediately after spinal cord injury (SCI). The purpose of our present study was to clarify the mechanism underlying how MR16-1 improves motor function after SCI. Quantitative analyses of inflammatory cells using flow cytometry, and immunohistochemistry with bone marrow-chimeric mice generated by transplanting genetically marked purified hematopoietic stem cells, revealed that MR16-1 dramatically switched the central player in the post-traumatic inflammation, from hematogenous macrophages to resident microglia. This change was accompanied by alterations in the expression of relevant cytokines within the injured spinal cord; the expression of recruiting chemokines including CCL2, CCL5, and CXCL10 was decreased, while that of Granulocyte/Macrophage-Colony Stimulating Factor (GM-CSF), a known mitogen for microglia, was increased. We also showed that the resident microglia expressed higher levels of phagocytic markers than the hematogenous macrophages. Consistent with these findings, we observed significantly decreased tissue damage and reduced levels of myelin debris and Nogo-A, the axonal growth inhibitor, by MR16-1 treatment. Moreover, we observed increased axonal regeneration and/or sprouting in the MR16-1-treated mice. Our findings indicate that the functional improvement elicited by MR16-1 involves microglial functions, and provide new insights into the role of IL-6 signaling in the pathology of SCI.