Osteoclast-Associated Receptor (OSCAR) Distribution in the Synovial Tissues of Patients with Active RA and TNF-α and RANKL Regulation of Expression by Osteoclasts In Vitro.

Osteoclast-associated receptor (OSCAR) is a co-stimulatory receptor in osteoclastogenesis. Synovial tissues from active rheumatoid arthritis (RA) patients express higher levels of OSCAR compared with osteoarthritic and normal patients; however, the comparison of OSCAR levels in different regions of active RA synovium has not been reported. The regulation of OSCAR by TNF-α and receptor activator of NF kappa ß ligand (RANKL) in pre-osteoclasts/osteoclasts in vitro is unclear. OSCAR and tartrate-resistant acid phosphatase (TRAP) expression levels did not differ between the cartilage pannus junction (CPJ) and non-CPJ regions in active RA. We demonstrate a similar pattern of OSCAR expression in the CPJ and non-CPJ synovial tissue from patients with active RA. OSCAR was associated with mononuclear cells in both the lining and sub-lining and endothelial cells (von Willebrand factor positive). Pre-osteoclasts (TRAP-positive cells) were present in the lining and sub-lining of both regions. OSCAR messenger RNA (mRNA) expression and release by pre-oscteoclasts/osteoclasts was modulated by RANKL with/without TNF-α in vitro. Osteoclast resorption on dentine slices was significantly greater with TNF-α pre-treatment and RANKL (10 ng/ml) than RANKL 10 or 50 ng/ml alone or RANKL 10 ng/ml with TNF-α given from day 3 post-RANKL. The lower levels of OSCAR mRNA expression corresponded with high osteoclast activity levels.

The X-Linked Inhibitor of Apoptosis Protein Inhibitor Embelin Suppresses Inflammation and Bone Erosion in Collagen Antibody Induced Arthritis Mice.

OBJECTIVE: To investigate the effect of Embelin, an inhibitor of X-Linked Inhibitor of Apoptosis Protein (XIAP), on inflammation and bone erosion in a collagen antibody induced arthritis (CAIA) in mice. METHODS: Four groups of mice (n = 6 per group) were allocated: CAIA untreated mice, CAIA treated with Prednisolone (10 mg/kg/day), CAIA treated with low dose Embelin (30 mg/kg/day), and CAIA treated with high dose Embelin (50 mg/kg/day). Joint inflammation was evaluated using clinical paw score and histological assessments. Bone erosion was assessed using micro-CT, tartrate resistant acid phosphatase (TRAP) staining, and serum carboxy-terminal collagen crosslinks (CTX-1) ELISA. Immunohistochemistry was used to detect XIAP protein. TUNEL was performed to identify apoptotic cells. RESULTS: Low dose, but not high dose Embelin, suppressed inflammation as reflected by lower paw scores (P < 0.05) and lower histological scores for inflammation. Low dose Embelin reduced serum CTX-1 (P < 0.05) and demonstrated lower histological score and TRAP counting, and slightly higher bone volume as compared to CAIA untreated mice. XIAP expression was not reduced but TUNEL positive cells were more abundant in Embelin treated CAIA mice. CONCLUSION: Low dose Embelin suppressed inflammation and serum CTX-1 in CAIA mice, indicating a potential use for Embelin to treat pathological bone loss.

Osteoimmunology: Major and Costimulatory Pathway Expression Associated with Chronic Inflammatory Induced Bone Loss.

The field of osteoimmunology has emerged in response to the range of evidences demonstrating the close interrelationship between the immune system and bone metabolism. This is pertinent to immune-mediated diseases, such as rheumatoid arthritis and periodontal disease, where there are chronic inflammation and local bone erosion. Periprosthetic osteolysis is another example of chronic inflammation with associated osteolysis. This may also involve immune mediation when occurring in a patient with rheumatoid arthritis (RA). Similarities in the regulation and mechanisms of bone loss are likely to be related to the inflammatory cytokines expressed in these diseases. This review highlights the role of immune-related factors influencing bone loss particularly in diseases of chronic inflammation where there is associated localized bone loss. The importance of the balance of the RANKL-RANK-OPG axis is discussed as well as the more recently appreciated role that receptors and adaptor proteins involved in the immunoreceptor tyrosine-based activation motif (ITAM) signaling pathway play. Although animal models are briefly discussed, the focus of this review is on the expression of ITAM associated molecules in relation to inflammation induced localized bone loss in RA, chronic periodontitis, and periprosthetic osteolysis, with an emphasis on the soluble and membrane bound factor osteoclast-associated receptor (OSCAR).

Parthenolide reduces empty lacunae and osteoclastic bone surface resorption induced by polyethylene particles in a murine calvarial model of peri-implant osteolysis.

The study aimed to determine the effects of parthenolide (PAR) on bone volume (BV) and bone surface resorption as assessed by live-animal microcomputed tomography (µCT) and possible osteocyte death as indicated by empty lacunae histologically in polyethylene (PE) particle-induced calvarial osteolysis in mice. Baseline µCT scans were conducted 7 days preimplantation of 2 × 10(8) PE particles/mL over the calvariae (day 0). PAR at 1 mg/kg/day was subcutaneously injected on days 0, 4, 7, and 10. At day 14, BV and surface resorption was analyzed with µCT. Calvarial tissue was processed for histomorphometric osteocyte evaluation. Serum was analyzed for type-1 carboxy-terminal collagen crosslinks (CTX-1) and osteoclast associated receptor (OSCAR) levels by ELISA. PE significantly decreased BV (p = 0.0368), increased surface bone resorption area (p = 0.0022), and increased the percentage of empty lacunae (p = 0.0043). Interestingly, PAR significantly reduced the resorption surface area (p = 0.0022) and the percentage of empty osteocyte lacunae (p = 0.0087) in the PE-calvariae, but it did not affect BV, serum CTX-1 or OSCAR levels. The ability of PAR to inhibit PE-induced surface bone erosion may better reflect the in vivo situation, where bone resorption occurs on the surface at the bone-implant interface and may also be related to the role of osteocytes in this pathology.

The immunoreceptor tyrosine-based activation motif (ITAM) -related factors are increased in synovial tissue and vasculature of rheumatoid arthritic joints.

INTRODUCTION: The immunoreceptor tyrosine-based activation motif (ITAM) pathway provides osteoclast co-stimulatory signals and regulates proliferation, survival and differentiation of effector immune cells. In the osteoclast, the receptors Triggering Receptor Expressed on Myeloid cells 2 (TREM2) and Osteoclast Associated Receptor (OSCAR) and their respective adaptor proteins, DAP12 and FcRγ mediate ITAM signals and induce calcium signaling and the crucial transcription factor, NFATc1. In rheumatoid arthritis (RA), OSCAR expression by monocytes is inversely correlated with disease activity. Additionally, serum levels of OSCAR are reduced in RA patients versus healthy controls suggesting that expression and secretion or cleavage of soluble (s) OSCAR is immune modulated. Recent data suggest that endothelial cells may also be a source of OSCAR. METHODS: ITAM receptors, their adaptor proteins, and NFATc1 and cathepsin K were detected in human synovial tissues by immunohistochemistry. Synovial tissues from patients with active RA were compared with tissue from patients in remission, osteoarthritis (OA) patients and healthy individuals. OSCAR was measured by immunoassay in synovial fluids recovered from active RA and OA patients. Endothelial cells were cultured with or without 5 ng/mL TNF-α or IL-1ß over 72 hours. Temporal expression of OSCAR mRNA was assessed by qRT PCR and OSCAR protein in the supernatant was measured by ELISA. RESULTS: Significantly higher (P < 0.05) NFATc1-positive inflammatory cell aggregates were found in active RA tissues than in healthy synovial tissue. Similarly, the percentage of OSCAR, FcRγ, DAP12 and TREM2 positive cells was significantly higher in active RA tissues compared to the healthy synovial tissue. Notably, OSCAR was strongly expressed in the microvasculature of the active RA tissues (9/9), inactive RA (8/9) weakly in OA (4/9) but only in the lumen of healthy synovial tissue (0/8). OSCAR levels were detected in synovial fluids from both RA (47 to 152 ng/mL) and OA (112 to 145 ng/mL) patients. Moreover, OSCAR mRNA expression and soluble OSCAR release was stimulated by TNF-α and IL1-ß in cultured endothelial cells. CONCLUSIONS: Increased levels of ITAM related factors were present in synovial tissue from active RA joints compared to OA and healthy joints. OSCAR was strongly expressed by the vasculature of active RA patients and membrane bound and soluble OSCAR was stimulated by inflammatory mediators in endothelial cells in vitro.

The expression of RANKL and OPG in the various grades of osteoarthritic cartilage.

The objective of the study was to determine whether cartilage expression of the bone regulating molecules receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) varies between the different grades of osteoarthritis (OA). Cartilage samples were obtained from 30 patients undergoing total hip/knee replacement surgery. Tissue sections were stained with Safranin O and graded. Immunohistochemical staining was then performed, and levels of RANKL and OPG expression were assessed using a semi-quantitative scoring system. In addition, levels of mRNA encoding for RANKL and OPG were determined by a relative real-time reverse transcription-polymerase chain reaction technique. We found that expression of RANKL protein, mRNA expression, and the ratio of RANKL: OPG mRNA was greater in grade 2 cartilage in comparison with grade 0 cartilage (P < 0.05). Increased RANKL staining in the grade 2 cartilage was predominantly in the peri-cellular region of the middle and deep zones as well as in the matrix of the superficial zone. OPG mRNA expression was greater in grade 3 cartilage in comparison with grade 0 cartilage (P < 0.05). Cartilage and subchondral bone are in close proximity and soluble proteins produced in the cartilage are likely to move from one compartment to the other. Our finding of increased expression of RANKL in grade 2 OA cartilage might explain the increase in bone turnover reported in the subchondral bone of OA patients. The changes seen in the different grades of tissue may also indicate that this effect occurs during the early stages of OA development.

TWEAK and Fn14 expression in the pathogenesis of joint inflammation and bone erosion in rheumatoid arthritis.

INTRODUCTION: TNF-like weak inducer of apoptosis (TWEAK) has been proposed as a mediator of inflammation and bone erosion in rheumatoid arthritis (RA). This study aimed to investigate TWEAK and TWEAK receptor (Fn14) expression in synovial tissue from patients with active and inactive rheumatoid arthritis (RA), osteoarthritis (OA) and normal controls and assess soluble (s)TWEAK levels in the synovial fluids from patients with active RA and OA. Effects of sTWEAK on osteoclasts and osteoblasts were investigated in vitro. METHODS: TWEAK and Fn14 expression were detected in synovial tissues by immunohistochemistry (IHC). Selected tissues were dual labelled with antibodies specific for TWEAK and lineage-selective cell surface markers CD68, Tryptase G, CD22 and CD38. TWEAK mRNA expression was examined in human peripheral blood mononuclear cells (PBMC) sorted on the basis of their expression of CD22. sTWEAK was detected in synovial fluid from OA and RA patients by ELISA. The effect of sTWEAK on PBMC and RAW 264.7 osteoclastogenesis was examined. The effect of sTWEAK on cell surface receptor activator of NF Kappa B Ligand (RANKL) expression by human osteoblasts was determined by flow cytometry. RESULTS: TWEAK and Fn14 expression were significantly higher in synovial tissue from all patient groups compared to the synovial tissue from control subjects (P < 0.05). TWEAK was significantly higher in active compared with inactive RA tissues (P < 0.05). TWEAK expression co-localised with a subset of CD38+ plasma cells and with CD22+ B-lymphocytes in RA tissues. Abundant TWEAK mRNA expression was detected in normal human CD22+ B cells. Higher levels of sTWEAK were observed in synovial fluids isolated from active RA compared with OA patients. sTWEAK did not stimulate osteoclast formation directly from PBMC, however, sTWEAK induced the surface expression of RANKL by human immature, STRO-1+ osteoblasts. CONCLUSIONS: The expression of TWEAK by CD22+ B cells and CD38+ plasma cells in RA synovium represents a novel potential pathogenic pathway. High levels of sTWEAK in active RA synovial fluid and of TWEAK and Fn14 in active RA tissue, together with the effect of TWEAK to induce osteoblastic RANKL expression, is consistent with TWEAK/Fn14 signalling being important in the pathogenesis of inflammation and bone erosion in RA.

Elevated expression of caspase-3 inhibitors, survivin and xIAP correlates with low levels of apoptosis in active rheumatoid synovium.

INTRODUCTION: Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a tumour necrosis factor (TNF) family member capable of inducing apoptosis in many cell types. METHODS: Using immunohistochemistry, terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) and real-time PCR we investigated the expression of TRAIL, TRAIL receptors and several key molecules of the intracellular apoptotic pathway in human synovial tissues from various types of arthritis and normal controls. Synovial tissues from patients with active rheumatoid arthritis (RA), inactive RA, osteoarthritis (OA) or spondyloarthritis (SpA) and normal individuals were studied. RESULTS: Significantly higher levels of TRAIL, TRAIL R1, TRAIL R2 and TRAIL R4 were observed in synovial tissues from patients with active RA compared with normal controls (p < 0.05). TRAIL, TRAIL R1 and TRAIL R4 were expressed by many of the cells expressing CD68 (macrophages). Lower levels of TUNEL but higher levels of cleaved caspase-3 staining were detected in tissue from active RA compared with inactive RA patients (p < 0.05). Higher levels of survivin and x-linked inhibitor of apoptosis protein (xIAP) were expressed in active RA synovial tissues compared with inactive RA observed at both the protein and mRNA levels. CONCLUSIONS: This study indicates that the induction of apoptosis in active RA synovial tissues is inhibited despite stimulation of the intracellular pathway(s) that lead to apoptosis. This inhibition of apoptosis was observed downstream of caspase-3 and may involve the caspase-3 inhibitors, survivin and xIAP.