Nobuo Koike

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.

Effect of 1,25-Dihydroxyvitamin D on Testicular Morphology and Gene Expression in Experimental Cryptorchid Mouse: Testis Specific cDNA Microarray Analysis and Potential Implication in Male Infertility

PURPOSE We evaluated the morphological effect and alterations in gene expression caused by 1,25-dihydroxyvitamin D treatment in the mouse testis undergoing experimental cryptorchidism and subsequent orchiopexy. MATERIALS AND METHODS The mean modified Johnsen score and testicular weight were estimated after 4 weeks of treatment with a 1,25-dihydroxyvitamin D prodrug. We examined sites of vitamin D receptor and mRNA expression, and 1,25-dihydroxyvitamin D analogue accumulation in the mouse testis. Also, we compared alterations in gene expression in the cryptorchid mouse testis with or without 1,25-dihydroxyvitamin D administration by testis specific cDNA microarray. We confirmed protein synthesis of a candidate among up-regulated genes in primary cultures of Sertolis cells by Western blotting. RESULTS Mean +/- SEM Johnsen score and testicular weight were increased by 1,25-dihydroxyvitamin D treatment but not significantly (6.12 +/- 0.33 vs 5.27 +/- 0.4 and 49.3 +/- 3.8 mg vs 42.6 +/- 5.5, p = 0.13 and 0.065, respectively). Vitamin D receptor and its mRNA were positive in Sertolis cells. The 1,25-dihydroxyvitamin D analogue accumulated mainly in Sertolis cells. Of 2,483 testis specific genes 19 showed up-regulation by 1,25-dihydroxyvitamin D treatment. Of these genes the regulator of cellular cholesterol homeostasis Abca1 was expressed mainly in Sertolis cells and influenced male fertility. In primary cultures of Sertolis cells the synthesis of Abca1 protein was increased by 1,25-dihydroxyvitamin D treatment but not by follicle-stimulating hormone or testosterone treatment. CONCLUSIONS We noted that 1,25-dihydroxyvitamin D contributes to spermatogenesis by up-regulating certain specific genes in Sertolis cells. Testis specific cDNA microarray analysis and vitamin D supplementation may have implications for managing male infertility.

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.

In Vivo Dose-related Receptor Binding of the Vitamin D Analogue [3H]-1,25-dihydroxy-22-oxavitamin D3 (OCT) in Rat Parathyroid, Kidney Distal and Proximal Tubules, Duodenum, and Skin, Studied by Quantitative Receptor Autoradiography

1,25-Dihydroxy-22-oxavitamin D3 (OCT) is a new synthetic analogue of 1,25(OH)2D3 with a low calcemic effect. This study utilized quantitative receptor autoradiography to determine the dose-related receptor binding and saturation among the vitamin D target cells: parathyroid chief cells, kidney distal and proximal tubule epithelium, duodenal absorptive epithelium, and epidermal keratinocytes. Rats were injected with 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, or 16.0 μg/kg bw of [26-3H]-OCT and sacrificed 1 hr afterwards. Then autoradiographs were prepared under identical conditions. In these target cells, nuclear uptake of radioactivity increased with dose and then achieved a plateau. However, their saturation doses showed differences: parathyroid chief cells 1-2 μg; duodenal absorptive epithelium, distal tubule epithelium, and epidermal keratinocytes 4-6 μg; proximal tubule epithelium 8 μg (per kg bw). In contrast, in nontarget cells, such as liver and duodenal smooth muscle, radioactivity did not concentrate in the nuclei but increased in the cytoplasm with dose, without plateauing. These results provide the first information on the relative saturabilities of various target cell populations with a vitamin D ligand. Parathyroid chief cells required the relatively lowest receptor saturation dose. This suggests a high sensitivity and response to OCT treatment with related therapeutic potential for the regulation of parathyroid function.

Distribution of 1,25-dihydroxyvitamin D3[22-oxa] in vivo receptor binding in adult and developing skin.

Because of the therapeutic potential of oxacalcitriol (OCT, 22-oxa-dihydroxyvitamin D3), in vivo studies were conducted in adult and neonatal rats to identify the nuclear receptor sites of action in different tissues of the skin. Results were compared with those for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and oestradiol from previous studies. Autoradiograms were prepared from the dorsal skin of adult rats and the skin of the leg and head regions of neonatal rats 1 or 2 h after the injection of3H-OCT. Specific nuclear concentrations of radioactivity, eliminated by competition with unlabelled OCT or 1,25(OH)2D3, were found in cells of the epidermis, outer hair sheath, hair bulb and sebaceous glands, but were absent or low in most fibroblasts of the dermis and hypodermis. The strongest nuclear binding of OCT was conspicuous in outer hair sheaths, where it was 1.5 to 3.2 times higher than in keratinocytes of the epidermis. The distribution of nuclear receptors for OCT was similar to that for 1,25(OH)2D3 but in part dissimilar to that for oestradiol. Oestradiol binding was found in the epidermis and hair sheaths, and also predominantly in fibroblasts of the dermis and hair dermal papillae. The results suggest genomic regulatory effects of OCT, similar to the effects of vitamin D, on proliferation, differentiation and activity of keratinocytes, growth and maintenance of hair, and proliferation and secretion of sebaceous glands. This may be utilized therapeutically, since OCT has a lower calcaemic effect than 1,25(OH)2D3.

Sustained Osteoblast Nuclear Receptor Binding of Converted 1α,25-Dihydroxyvitamin D3 after Administration of 3H-1α-Hydroxyvitamin D3: A Combined Receptor Autoradiography and Radioassay Time Course Study with Comparison to 3H-1α,25-Dihydroxyvitamin D3

Abstract. The present study was undertaken to clarify the receptor distribution and the pharmacokinetics of 3H-1α(OH)D3, and 3H-1α,25(OH)2D3 for comparison. Receptor autoradiography was used after intravenous injection to 3-day-old neonatal rats and radioassay-HPLC after oral application to young adult rats. Corresponding results were obtained from both receptor autoradiography and radioassay. After 3H-1α(OH)D3 administration, uptake was delayed but sustained over a long period of time and the concentration of silver grains (autoradiography) or recovered 3H-1α,25(OH)2D3 (radioassay) peaked at a lower level. After 3H-1α,25(OH)2D3 administration, osteoblast nuclear, whole bone uptake and retention of radiolabeled compound were relatively rapid and short in duration. Nuclear uptake in osteoblasts after administration of 3H-1α(OH)D3 was abolished in competition studies with 10-fold unlabeled 1α,25(OH)2D3. These results indicate that 1α(OH)D3 continuously supplies osteoblasts with converted 1α,25(OH)2D3 and would not spread to the cells because of the low binding affinity of the receptor. Accordingly, 1α(OH)D3 appears to have some therapeutic properties different from 1α,25(OH)2D3 because of a relatively slow and sustained accumulation of the receptor and less Cmax (pharmacokinetics) compared with 1α,25(OH)2D3.

Anemia in monkey collagen-induced arthritis is correlated with serum IL-6, but not TNFα

We characterized the anemia in monkey collagen-induced arthritis (CIA) to evaluate whether this model is useful to analyze the basis of an anemia of inflammatory diseases. Cynomolgus monkey was immunized with bovine type II collagen on days 0 and 21. Blood samples were collected regularly and hematological parameters, biochemical parameters and cytokine levels were monitored. Red blood cell (RBC) counts, hematocrit (Ht), and hemoglobin (Hb) gradually decreased after immunization and reached the bottom on day 35. CRP rose rapidly after first immunization and reached a peak on day 21. Serum iron levels and transferrin (Tf) saturation were dropped after immunization and reached a bottom on day 28. Thereafter it returned to normal. On the other hand, ferritin levels increased after immunization. IL-6 levels showed positive correlation with CRP, and negative correlation with Hb, RBC counts and serum iron, but TNFα did not show any correlation. In conclusion, the anemia in monkey CIA is very similar to human anemia of inflammatory diseases concerning the changes of serum parameters. And our data strongly suggest that IL-6 is an essential cytokine for the development of the anemia in monkey CIA.

Combination treatment with eldecalcitol (ED-71) and raloxifene improves bone mechanical strength by suppressing bone turnover and increasing bone mineral density in ovariectomized rats.

The aim of this study was to investigate the effect of combination treatment with eldecalcitol (ELD) and raloxifene (RAL) on bone turnover, bone mineral density (BMD), and bone strength. Eight-month-old rats were ovariectomized (OVX) or sham operated, and divided into five groups (Sham, OVX+vehicle, OVX+RAL, OVX+ELD and OVX+ELD+RAL). ELD (7.5 ng/kg) and RAL (0.3mg/kg) were orally administered alone or in combination daily. Urinary deoxypyridinoline (DPD) levels were measured after 4, 8, and 12 weeks of treatment. After 12 weeks of treatment, BMD and mechanical properties of the lumbar spine and femur were assessed, and bone histomorphometry was performed. Urinary DPD levels in all the treatment groups were significantly decreased compared with the OVX+vehicle group. At 4 weeks of treatment, urinary DPD level of the combination group was significantly lower than that of either monotherapy group. The reduction in the BMD of the lumbar spine and femur by OVX was significantly prevented in all the treatment groups, and the BMD in the combination group was significantly higher than that in either monotherapy group. The ultimate load and work to failure of the fifth lumbar vertebra were significantly improved only by the combination treatment. The femoral midshaft ultimate load was significantly increased in the OVX+ELD group and the combination group, and the femoral midshaft work to failure was increased only in the combination group. Bone histomorphometric analysis using the third lumbar vertebra revealed that osteoblast surface (Ob.S/BS), osteoclast surface (Oc.S/BS) and osteoclast number (N.Oc/BS) significantly decreased in all treatment groups, and osteoid surface (OS/BS) and bone formation rate (BFR/BS) significantly decreased in the ELD-treated and combination groups. The values of Ob.S/BS and OS/BS in the combination group were lower than those in either of the monotherapy groups. The bone formation parameters in the combination group were not reduced to below levels of the sham-operated control, suggesting that the combination therapy with ELD and RAL may not cause oversuppression of bone turnover. These results indicated that the combination treatment with ELD and RAL might be a beneficial therapy with respect to their combined effects of enhancing the mechanical properties of trabecular and cortical bone by suppressing bone turnover and increasing BMD more than either monotherapy.