Shigeru Kotake

IL-17 in synovial fluids from patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis

IL-17 is a newly discovered T cell-derived cytokine whose role in osteoclast development has not been fully elucidated. Treatment of cocultures of mouse hemopoietic cells and primary osteoblasts with recombinant human IL-17 induced the formation of multinucleated cells, which satisfied major criteria of osteoclasts, including tartrate-resistant acid phosphatase activity, calcitonin receptors, and pit formation on dentine slices. Direct interaction between osteoclast progenitors and osteoblasts was required for IL-17-induced osteoclastogenesis, which was completely inhibited by adding indomethacin or NS398, a selective inhibitor of cyclooxgenase-2 (COX-2). Adding IL-17 increased prostaglandin E2 (PGE2) synthesis in cocultures of bone marrow cells and osteoblasts and in single cultures of osteoblasts, but not in single cultures of bone marrow cells. In addition, IL-17 dose-dependently induced expression of osteoclast differentiation factor (ODF) mRNA in osteoblasts. ODF is a membrane-associated protein that transduces an essential signal(s) to osteoclast progenitors for differentiation into osteoclasts. Osteoclastogenesis inhibitory factor (OCIF), a decoy receptor of ODF, completely inhibited IL-17-induced osteoclast differentiation in the cocultures. Levels of IL-17 in synovial fluids were significantly higher in rheumatoid arthritis (RA) patients than osteoarthritis (OA) patients. Anti-IL-17 antibody significantly inhibited osteoclast formation induced by culture media of RA synovial tissues. These findings suggest that IL-17 first acts on osteoblasts, which stimulates both COX-2-dependent PGE2 synthesis and ODF gene expression, which in turn induce differentiation of osteoclast progenitors into mature osteoclasts, and that IL-17 is a crucial cytokine for osteoclastic bone resorption in RA patients.

IFN-γ-producing human T cells directly induce osteoclastogenesis from human monocytes via the expression of RANKL

The current study explored our hypothesis that IFN‐γ‐producing human T cells inhibit human osteoclast formation. Activated T cells derived from human PBMC were divided into IFN‐γ‐producing T cells (IFN‐γ+ T cells) and IFN‐γ‐non‐producing T cells (IFN‐γ– T cells). IFN‐γ+ T cells were cultured with human monocytes in the presence of macrophage‐CSF alone. The concentration of soluble receptor activator of NF‐κB ligand (RANKL) and IFN‐γ, and the amount of membrane type RANKL expressed on T cells, were measured by ELISA. In the patients with early rheumatoid arthritis (RA) treated with non‐steroidal anti‐inflammatory drugs alone, CD4+ T cells expressing both IFN‐γ and RANKL were detected by flow cytometry. Surprisingly, IFN‐γ+ T cells, but not IFN‐γ– T cells, induced osteoclastogenesis from monocytes, which was completely inhibited by adding osteoprotegerin and increased by adding anti‐IFN‐γ antibodies. The levels of both soluble and membrane type RANKL were elevated in IFN‐γ+ T cells. The ratio of CD4+ T cells expressing both IFN‐γ and RANKL in total CD4+ T cells from PBMC was elevated in RA patients. Contrary to our hypothesis, IFN‐γ+ human T cells induced osteoclastogenesis through the expression of RANKL, suggesting that Th1 cells play a direct role in bone resorption in Th1 dominant diseases such as RA.

Effects of recombinant human bone morphogenetic protein-2 on human bone marrow cells cultured with various biomaterials.

Recombinant human bone morphogenetic protein-2 (rhBMP-2) is known to induce orthotopic and ectopic bone formation in vivo. Several in vitro studies using rat or mouse clonal cell lines have shown that rhBMP-2 may be involved in the differentiation of osteoblasts from osteoblast precursor cells or stromal cells in the bone marrow. However, there is little information available about the effects of rhBMP-2 on cultured human bone marrow cells. We investigated the effects of rhBMP-2 cultured on human bone marrow cells and osteoblastic cells on various biomaterials. Human bone cells were divided into fresh bone marrow cells, fibroblast colony-forming units (cfu-F, stromal precursors), and osteoblastic cells. The cells were cultured with or without rhBMP-2 on various biomaterials, including titanium alloy, pure titanium, cobalt alloy, and hydroxyapatite. It was found that rhBMP-2 (500 ng/mL) significantly stimulated alkaline phosphatase production by fresh bone marrow cells and cfu-F. However, when cultured on titanium alloy or pure titanium, only fresh bone marrow cells showed an increase of alkaline phosphatase production after rhBMP-2 stimulation. Production of osteocalcin, a marker of mature osteoblasts, was not stimulated by rhBMP-2 in any combinations tested. These findings suggest that rhBMP-2 may be involved in inducing the differentiation of osteoblast precursor cells into osteoblastic cells rather than stimulating further differentiation of osteoblastic cells into mature osteoblasts. In addition, grafts of fresh human bone marrow cells of cfu-F stimulated by rhBMP-2 may have the potential to promote bone formation at sites of nonunion as well as around titanium joint prostheses.

Stromal cell activity in bone marrow from the tibia and iliac crest of patients with rheumatoid arthritis

Abstract Bone marrow aspirates were obtained from the iliac crest and tibial epiphysis in 23 patients with rheumatoid arthritis (RA) who were undergoing total knee arthroplasty. The number of fibroblast colony-forming units (CFU-F), which contain osteogenic precursor cells, and alkaline phosphatase (ALP) activity, as a marker of the osteoblastic phenotype, were compared between the iliac and tibial marrow for each patient. The prevalence of CFU-F in tibial marrow was similar to that in iliac marrow (96% vs 100%, respectively). However, the average number of CFU-F per 4 × 105 bone marrow mononuclear cells was significantly lower in tibial marrow than in iliac marrow (8.2 vs 28.1, respectively; P < 0.01). Although ALP activity was detected in all iliac and tibial marrow specimens, it was significantly lower in tibial marrow compared with iliac marrow (3.7 vs 11.9 nmol/min/mg protein, respectively; P < 0.01). In addition, there was a significant correlation between the patients age and the number of CFU-F in iliac marrow (r = −0.547; P < 0.01), although there was no correlation in tibial marrow. These results demonstrate that the osteogenic activity of bone marrow varies at different sites in patients with RA. The data may also contribute to further investigation into the differential effects of various disease processes on systemic as well as local stromal cell activity in bone marrow.

Associations between methotrexate treatment and methylenetetrahydrofolate reductase gene polymorphisms with incident fractures in Japanese female rheumatoid arthritis patients.

Several case reports have described associations between pathological nonvertebral fractures and low-dose methotrexate (MTX) in rheumatoid arthritis (RA) patients. Furthermore, a significant association between the C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene and incident fractures has been reported in postmenopausal women. We attempted to determine whether MTX use and MTHFR polymorphisms are associated with incident fracture risk in Japanese female RA patients. DNA samples, laboratory data, and clinical data were obtained from 731 female RA patients more than 50xa0years old as part of the Institute of Rheumatology Rheumatoid Arthritis (IORRA) observational cohort study. Genotyping of the MTHFR polymorphisms C677T and A1298C was performed using TaqMan SNP Genotyping Assays. MTX use, MTHFR polymorphisms, and other potential risk factors predictive of fracture were analyzed by Cox proportional hazards regression models, including time-dependent covariates. During 78xa0months from October 2000 to March 2007, 25 and 90 patients developed vertebral and nonvertebral fractures, respectively. Patients with nonvertebral fractures were more likely to take MTX (Pxa0=xa00.011; odds ratio, 1.77; 95% confidence interval, 1.13–2.76) compared to patients without fractures. Although the C677T and A1298C polymorphisms were not significantly associated with incident fracture risk, MTX use, age, disease duration, and Japanese health assessment questionnaire score were significantly (Pxa0<xa00.05) and independently associated with nonvertebral fracture incidence. Our results suggest that MTX use is associated with a nonvertebral fracture risk, whereas MTHFR polymorphism status does not appear to be a clinically useful marker for predicting fracture risk in Japanese female RA patients.

T-cell leukemia translocation-associated gene (TCTA) protein is required for human osteoclastogenesis☆

Synovial tissues of patients with rheumatoid arthritis (RA) include factors regulating bone resorption, such as receptor activator NF-kappaB ligand (RANKL), TNFalpha, IL-6, IL-17 and IFNgamma. However, in addition to these cytokines, other factors expressed in synovial tissues may play a role in resorbing bone. Here, our objective was to identify novel proteins expressed in synovial tissues of RA that regulate human osteoclastogenesis. Proteins were purified from synovial tissues of patients with RA, using gel filtration chromatography, ion-exchange chromatography, reverse-aspect HPLC, and mass spectrometry. We evaluated the effects of the purified fractions on human osteoclastogenesis induced by RANKL and M-CSF. We determined the amino acid sequences showing inhibitory activity on human osteoclastogenesis. In addition, we synthesized novel peptides from the molecule including the amino acid sequences. Then, we evaluated the effects of the peptides and antibodies against the molecule on human osteoclastogenesis from monocytes and mature osteoclasts, and on pit formation by mature osteoclasts using Osteologic discs. We examined the effect of the peptide on the expression of both mRNA and protein of NFATc1. We also examined the effect of RANKL on the expression of mRNA of the molecule on osteoclasts and macrophages. We identified a small peptide including Gly-Gln-Asn (GQN) with inhibitory activity on human osteoclastogenesis. We then found that GQN is included in the amino acid sequence of the extra-cellular domain of TCTA protein, which is expressed ubiquitously in normal human tissues, but whose function has not been clarified. We designed novel peptides, including GQN, from the sequence of TCTA protein. One of these peptides (29-mer), but not a scrambled peptide for the 29-mer peptide, potently inhibited RANKL-induced human osteoclastogenesis. The peptide also inhibited pit formation of mature human osteoclasts and suppressed the formation of large osteoclasts in the culture of mature osteoclasts. Furthermore, polyclonal antibodies against TCTA protein suppressed the formation of large osteoclasts in the cultures of both monocytes and mature osteoclasts, supporting our hypothesis. Peptide A did not significantly inhibit the expression of both mRNA and protein of NFATc1 in osteoclasts. Our novel peptide and polyclonal antibodies against the peptide inhibited human osteoclastogenesis and the function of mature osteoclasts, preventing cellular fusion by TCTA protein and a putative counterpart molecule.

Adrenomedullin in synovial fluids from patients with rheumatoid arthritis inhibits interleukin 6 production from synoviocytes

A drenomedullin (AM) is a hypotensive peptide found in human pheochromocytoma tissue, which comprises 52 amino acids with an intramolecular disulphide bond.1,2 The ring structure and amidated C-terminus of AM are critical for its receptor binding and hypotensive activity. The mature AM is synthesised as glycine extended AM followed by C-terminal amidation to assume a biologically active form in tissues. AM has a vasorelaxant effect, antagonising the vasospastic effect of endothelin-1 (ET-1). Recently, proinflammatory cytokines, such as tumour necrosis factors α (TNFα) and interleukin-1 (IL1), were found to stimulate production and secretion of AM from vascular endothelial cells and vascular smooth muscle cells in vitro, suggesting that AM interacts with the immune system.3 However, AM reduces the production of TNFα from macrophages stimulated with lipopolysaccharide. In addition, AM shows an anti-inflammatory effect that reduces the production of …

A1330V polymorphism of low-density lipoprotein receptor-related protein 5 gene and self-reported incident fractures in Japanese female patients with rheumatoid arthritis

We attempted to determine whether the A1330V polymorphism of the low-density lipoprotein receptor-related protein 5 (LRP5) gene is associated with a risk of self-reported incident fractures and hypercholesterolemia in Japanese patients with rheumatoid arthritis (RA). DNA samples, laboratory data, and clinical data were obtained from 563 female RA patients who participated in the Institute of Rheumatology Rheumatoid Arthritis (IORRA) observational cohort study. A1330V genotyping was performed using a custom TaqMan assay. Multiple logistic regression analyses showed that any incident fracture was significantly associated with older age (Pxa0=xa00.000000036), high Japanese Health Assessment Questionnaire (J-HAQ) score (Pxa0=xa00.016), and high daily prednisolone dose (Pxa0=xa00.031), but not with the A1330V polymorphism, while serum total cholesterol levels ≥220xa0mg/100xa0mL were independently correlated with baseline older age (Pxa0=xa00.00011), low J-HAQ score (Pxa0=xa00.0098), high body mass index (Pxa0=xa00.024), 1330VV genotype (Pxa0=xa00.027), and high daily prednisolone dose (Pxa0=xa00.031). Our results suggest that this LPR5 polymorphism does not appear to be a clinically useful marker for the prediction of fracture risk in Japanese female RA patients, although it is associated with increased serum total cholesterol levels.

Effect of bone marrow grafting on the titanium porous-coated implant in bilateral total knee arthroplasty.

Background and purposeu2003Poor bone ingrowth into the porous coating of tibial components has been reported. We hypothesized that iliac marrow grafting might be useful to enhance bone ingrowth into a porous-coated implant. The first part of this study was to examine the presence of fibroblast colony-forming units (CFUF) containing osteogenic precursor cells in tibial bone marrow and iliac bone marrow. The second aim was to compare the clinical and radiographic results after bilateral total knee arthroplasty (TKA) with and without autologous bone marrow transplantation to the bone-implant interface. Methodsu2003Simultaneous bilateral TKA was performed in 21 patients with osteoarthritis. Aspirated iliac bone marrow was transplanted to the interface of one randomly selected porous-coated tibial component in each patient, and contralateral knees served as controls. All of the 21 patients were followed for 5 years. Resultsu2003The average number of CFU-F was significantly lower in tibial marrow than in iliac marrow (p = 0.008). The final fluoroscopically-guided radiographs revealed a decrease in the number of knees with radiolucent lines after marrow grafting compared to those without grafting (p = 0.004). Interpretationu2003Iliac bone marrow is useful as a bone grafting material to enhance the biological fixation in porous-coated implants.

Raised plasma adrenomedullin level in Behçet's disease patients

Abstract To investigate the role of plasma adrenomedullin (AM) in the pathogenesis of Behçets disease (BD) patients with inactive ocular complications or ocular attack, 18 consecutive BD patients with ocular complications, including 1 BD patient with ocular attack, another group of 6 BD patients with ocular attack, and 10 normal volunteers were evaluated. All BD patients were regularly followed at ophthalmic outpatient clinics. Levels of both total and mature AM in plasma were measured by immunoradiometric assay. Plasma levels of endothelin-1 (ET-1) were also measured by radioimmunoassay. We also measured the levels of C-reactive protein (CRP) in plasma. Levels of total AM in plasma (mean ± SD, 19.6 ± 6.9u2009fmol/l) were significantly higher in BD patients than in normal volunteers (14.5 ± 3.6u2009fmol/l) (P = 0.01). The levels of mature AM were also higher in BD patients (1.6 ± 0.4u2009fmol/l) than in normal volunteers (0.3 ± 0.6u2009fmol/l) (P = 0.002). The levels of AM in patients with ocular attack were higher than those in normal volunteers, although there was no significant difference compared to levels of AM in BD patients without ocular attack. AM may play an important role as an antiinflammation factor or may reflect endothelial damage as a marker of disease activity in BD patients.