High levels of osteoprotegerin and soluble receptor activator of nuclear factor κB ligand in serum of rheumatoid arthritis patients and their normalization after anti–tumor necrosis factor α treatment

Objective

To test the hypotheses that 1) proinflammatory cytokines affect osteoprotegerin (OPG) and soluble receptor activator of nuclear factor κB ligand (sRANKL) production and therefore the OPG and sRANKL levels differ in rheumatoid arthritis (RA) patients in comparison with healthy individuals; and 2) anti–tumor necrosis factor α (anti–TNFα) therapy influences OPG and sRANKL levels.

Methods

Sera were obtained from healthy individuals or RA patients receiving the combination of infliximab and methotrexate. Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were isolated from RA patients. Fibroblast‐like synoviocytes (FLS) were isolated from synovial tissue obtained at total knee replacement in RA patients. Supernatants from cells stimulated with cytokines were collected after culture in vitro. Concentrations of OPG and sRANKL were determined by enzyme‐linked immunosorbent assays.

Results

A strong positive correlation between OPG concentration and age was observed in healthy individuals but not in RA patients. The OPG and sRANKL levels were higher in RA patients than in healthy controls. Cultured FLS spontaneously secreted much higher amounts of OPG than PBMCs or SFMCs. Proinflammatory cytokines enhanced OPG production. Anti‐TNFα treatment resulted in the normalization of serum OPG and sRANKL levels in RA patients without influencing the OPG:sRANKL ratio.

Conclusion

Although higher serum levels of OPG and sRANKL are present in RA patients than in healthy individuals, the ratio of OPG:sRANKL is similar. There is an age‐dependent increase of OPG but not sRANKL levels in healthy subjects. Anti‐TNFα treatment results in the normalization of elevated levels of OPG and sRANKL in RA patients.

     

Role of osteoprotegerin/receptor activator of nuclear factor kappa B/receptor activator of nuclear factor kappa B ligand axis in nonalcoholic fatty liver disease

Concomitantly with the increase in the prevalences of overweight/obesity, nonalcoholic fatty liver disease(NAFLD) has worldwide become the main cause of chronic liver disease in both adults and children. Patients with fatty liver display features of metabolic syndrome(Met S), like insulin resistance(IR), glucose intolerance, hypertension and dyslipidemia. Recently, epidemiological studies have linked obesity, Met S, and NAFLD to decreased bone mineral density and osteoporosis, highlighting an intricate interplay among bone, adipose tissue, and liver. Osteoprotegerin(OPG), an important symbol of the receptor activator of nuclear factor-B ligand/receptor activator of nuclear factor kappa B/OPG system activation, typically considered for its role in bone metabolism, may also play critical roles in the initiation and perpetuation of obesityrelated comorbidities. Clinical data have indicated that OPG concentrations are associated with hypertension, left ventricular hypertrophy, vascular calcification, endothelial dysfunction, and severity of liver damage in chronic hepatitis C. Nonetheless, the relationship between circulating OPG and IR as a key feature of Met S as well as between OPG and NAFLD remains uncertain. Thus, the aims of the present review are to provide the existent knowledge on these associations and to discuss briefly the underlying mechanisms linking OPG and NAFLD.     

Methylation of receptor activator of nuclear factor kappa ligand (RANKL) gene in rheumatoid arthritis patients

BackgroundRheumatoid arthritis (RA) is an autoimmune inflammatory disease characterized by synovitis, cartilage damage and bone resorption. Methylation of deoxyribonucleic acid plays a crucial role in repressing gene expression. Receptor activator of nuclear factor-kappa ligand (RANKL) controls bone homeostasis.Aim of the workTo assess the serum level of RANKL and its gene promoter methylation in RA patients and to determine its association with clinical characteristics and disease activity.Patients and methodsThe study included 40 RA patients and 40 control. The disease activity score (DAS28) was assessed. Frequency of RANKL gene promoter methylation was determined by quantitative methylation specific PCR (QMSP) and serum RANKL level by enzyme linked immunosorbant assay (ELISA).ResultsPatients mean age was 46.8 ± 10.6 years, 36 females and 4 males (F:M 9:1) with median disease duration 4.5 years. Positive rheumatoid factor, anti-cyclic citrullinated peptide and C-reactive protein were present in 65 %, 75 % and 55 % of cases. Methylation percentage of RANKL gene promoter was significantly lower in patients (3.4 %) than in controls (3.7 %)(p = 0.035) while serum level was significantly increased in patients (9.1 ng/ml,5.3–11.8 ng/ml) than in controls (5.7 ng/ml, 4.5–8 ng/ml)(p = 0.003). RANKL methylation frequency was inversely associated with serum level (rs = -0.21,p = 0.06). There was no significant correlation of RANKL serum level and methylation with DAS28 (r = 0.03,p = 0.87 and r = 0.06,p = 0.73 respectively). RANKL serum level (>9.5 ng/ml) and methylation percentage (≤9%) significantly discriminate RA cases from control (sensitivity 47.5 %, specificity 91.9 %; p = 0.001 and sensitivity 100 %, specificity 40 %; p = 0.03 respectively).ConclusionRA patients expressed elevated serum RANKL with low methylation.     

Endurance running acutely raises plasma osteoprotegerin and lowers plasma receptor activator of nuclear factor kappa B ligand

Abstract The balance of the 2 cytokines, osteoprotegerin (OPG) and the receptor activator of nuclear factor kappa B ligand (soluble (s)RANKL), is known to have considerable influence on bone formation and degradation. Plasma concentrations of OPG and (s)RANKL were determined in a total of 31 long-distance runners before and immediately after running distances of either 15 or 42.195 km, respectively. In both groups of endurance runners, a significant decrease of sRANKL was observed during the run, the extent of which correlated to the running distance. Furthermore, OPG increased only in runners covering the marathon distance of 42.195 km. We hypothesize that the known positive effect of long-distance running on the skeletal mass may be mediated by the OPG/sRANKL system.     

Receptor activator of nuclear factor kappa B ligand serum and synovial fluid level. A comparative study between rheumatoid arthritis and osteoarthritis

To assess the levels of receptor activator of NF-κB ligand (RANKL) in serum and synovial fluid (SF) of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to correlate its levels with disease activity and severity. Serum and SF levels of RANKL were measured in 24 patients with RA (Group I) and 20 patients with OA (Group II); patients were selected according to the ACR criteria, and serum RANKL was measured in 13 healthy controls. All patients were subjected to full rheumatological assessment. In RA group, serum level of RANKL was significantly higher than control group (P?=?0.01), but not correlated with disease activity and severity parameters apart from number of tender joints (P?=?0.03). SF level of RANKL was significantly correlated with disease duration (P?=?0.02), number of tender (P?=?0.002) and swollen joints (P?=?003), ESR (P?=?0.01), CRP (P?=?0.000), DAS-28 (P?=?0.004), and SENS (P?=?0.03). In patients with OA, serum level of RANKL was significantly higher than the control group (P?<?0.001), and it was statistically insignificant with clinical, laboratory, or radiological data, while SF level of RANKL was statistically significantly higher in patients with Heberden and Bouchard nodes (P?=?0.007), Kellgren—Lawrence score (P?=?0.002), and with the erosive changes of hands (P?=?0.006). The mean serum RANKL in RA group was insignificant with that of total OA group. SF level of RANKL was significantly higher in RA than erosive OA patients and in erosive than non-erosive OA with (P?=?0.001, in each one). The SF level of RANKL is an important marker of both disease activity and severity in RA patients; while in OA patients, it is an important marker of disease severity especially in erosive than non-erosive types. Serum level of RANKL may be of low benefit in disease activity and severity of both rheumatoid arthritis and osteoarthritis.     

Influence of receptor activator of nuclear factor kappa B ligand,osteoprotegerin and interleukin-33 on bone metabolism in patients with long-standing ulcerative colitis

BackgroundUlcerative colitis (UC) is a chronic disease with periods of remission and recurrences. Dysfunction of the local immune response leads to chronic inflammation within the large intestine which triggers morphological changes in the intestinal wall as well as induces the synthesis of numerous factors that have an adverse impact on the bone metabolism.The aim of the study was to determine the expression of RANKL, OPG and IL-33 in mucosal biopsies of UC patients with long disease duration as well as serum level of these cytokines in the context of bone density and bone metabolism.Materials and methodsThe UC group consisted of 56 patients with average disease duration of 16 y. The control group comprised 37 healthy individuals. Local expression of cytokines was assessed in the biopsies of colonic mucosa by the real-time PCR and immunohistochemistry (IHC), and their serum concentration was measured by ELISA.ResultsThe increased bone resorption observed in patients with UC was reflected by low bone density and high serum level of C-terminal telopeptide (CTX). Mucosal RANKL expression and serum concentration were similar in UC group and healthy subjects, however, UC patients had higher local expression of OPG and serum OPG concentration. Increased IL-33 gene expression was observed only in UC at the mRNA level. We propose that bone resorption in UC patients despite OPG up-regulation could be caused by IL-33-induced mucosal synthesis of a potent proinflammatory cytokine, such as TNF-α, known as a possible inducer of osteoclastogenesis in the way independent of RANKL.     

IL-7 induces bone loss in vivo by induction of receptor activator of nuclear factor kappa B ligand and tumor necrosis factor alpha from T cells

IL-7, a powerful lymphopoietic cytokine, is elevated in rheumatoid arthritis (RA) and known to induce bone loss when administered in vivo. IL-7 has been suggested to induce bone loss, in part, by stimulating the proliferation of B220(+) cells, a population capable of acting as early osteoclast (OC) precursors. However, the mechanism by which IL-7 leads to differentiation of precursors into mature OCs remains unknown. We previously reported that, in vitro, IL-7 up-regulated T cell cytokines including receptor activator of nuclear factor kappaB ligand (RANKL). To demonstrate the importance of T cells to the bone-wasting effect of IL-7 in vivo, we have now examined IL-7-induced bone loss in T cell-deficient nude mice. We show that T cell-replete mice undergo significant osteoclastic bone loss after IL-7 administration, concurrent with induction of RANKL and tumor necrosis factor alpha (TNF-alpha) secretion by splenic T cells. In contrast, nude mice were resistant to IL-7-induced bone loss and showed no detectable increase in either RANKL or TNF-alpha, despite an up-regulation of B220(+) cells. Importantly, T cell adoptive transfer into nude mice restored IL-7-induced bone loss, and RANKL and TNF-alpha secretion, demonstrating that T cells are essential mediators of IL-7-induced bone loss in vivo.     

Systemic anti-tumor necrosis factor alpha therapy in rheumatoid arthritis down-regulates synovial tumor necrosis factor alpha synthesis

OBJECTIVE: To investigate the hypothesis that tumor necrosis factor alpha (TNFalpha) blockade in rheumatoid arthritis (RA) diminishes synovial synthesis of TNFalpha, interleukin-1alpha (IL-1alpha), and IL-1beta. METHODS: Patients with active RA received a single 10 mg/kg infusion of infliximab. Multiple synovial biopsy specimens were obtained from a knee the day before infusion and 14 days later. A modified immunohistochemical method detecting cytokine-producing rather than cytokine-binding cells was applied to determine synthesis of TNFalpha, IL-1alpha, and IL-1beta in fixed, cryopreserved sections. Computerized image analysis using two different methodologies was performed by independent observers blinded to the identity of samples. RESULTS: All 8 patients met the American College of Rheumatology 20% improvement response criteria (ACR 20) at 2 weeks, and half of these patients met the ACR 50. With a few exceptions, there was concordance between both image analysis methodologies regarding the direction of change in immunopositive area fraction for all cytokines analyzed. TNFalpha synthesis was significantly reduced after treatment (P = 0.05 at the Karolinska Institute, Stockholm, Sweden; P = 0.008 at the Kennedy Institute, London, UK). Patients meeting the ACR 50 were those with the highest baseline levels of TNFalpha synthesis. There was a significant correlation between baseline levels of TNFalpha expression and change in TNFalpha levels in response to therapy. Both IL-1alpha and IL-1beta synthesis were reduced in 3 patients; IL-1alpha synthesis alone was reduced in 2 patients and IL-1beta synthesis alone was reduced in 2 patients. In 1 patient, neither IL-1alpha nor IL-1beta synthesis was reduced. CONCLUSION: Analysis of synovial tissue by means of immunomorphology and image analysis in a clinical trial setting may allow the drawing of biologically meaningful conclusions. Synovial TNFalpha synthesis was reduced 2 weeks after infliximab treatment. Reductions in IL-1alpha and IL-1beta synthesis were demonstrated in a subgroup of patients. High levels of synovial TNFalpha production prior to treatment may predict responsiveness to therapy.     

Tumour necrosis factor-related apoptosis-inducing ligand and osteoprotegerin serum levels in psoriatic arthritis

OBJECTIVES: The degree of bone loss in patients with psoriatic arthritis (PsA) has not been well-defined. We tested the hypothesis, whether serum levels of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), a pro-apoptotic cytokine and osteoprotegerin (OPG), an anti-osteoclastic cytokine, are associated with changes in biochemical markers of bone turnover or bone mineral density (BMD) in patients with PsA. METHODS: In a cross-sectional study, we evaluated biochemical markers of bone turnover, BMD and serum levels of TRAIL and OPG in 116 patients with PsA (mean age: 52+/-13 yrs). RESULTS: In patients with PsA, osteopenia was present in one-third of women and men, while osteoporosis was more frequent in men (10.2%) than in women (1.75%). Serum levels of TRAIL were significantly higher in patients with PsA (66.1+/-45.3 pmol/l) compared with controls (50.0+/-20.1 pmol/l, P<0.01), whereas OPG serum levels were not different. There were no associations between TRAIL or OPG serum levels with BMD and biochemical markers of bone turnover. However, TRAIL serum levels were associated with C-reactive protein (CRP) levels (R = 0.201, P<0.05), whereas OPG serum levels were associated with the erythrocyte sedimentation rate (R=0.215, P<0.05). CONCLUSION: In summary, BMD is decreased in one-third of patients with PsA, and predominantly men with PsA suffer from osteoporosis. While TRAIL serum levels are increased in PsA and correlated with CRP levels, neither TRAIL nor OPG serum levels are correlated with BMD or markers of bone metabolism.     

Reduction of urinary levels of pyridinoline and deoxypyridinoline and serum levels of soluble receptor activator of NF-kappaB ligand by etanercept in patients with rheumatoid arthritis

The effects of soluble TNF-α receptor, etanercept, on bone metabolism were investigated in patients with rheumatoid arthritis (RA). Thirty RA patients were administered etanercept once or twice a week for more than 6 months. We evaluated clinical and laboratory parameters and measured urinary excretion levels of pyridinoline (PYD), deoxypyridinoline (DPD), cross-linked N-telopeptides of type I collagen (NTX), and serum levels of bone alkaline phosphatase (BAP), osteoprotegerin (OPG), and soluble receptor activator of NFκB ligand (sRANKL) at the baseline and at 3 and 6 months after initial treatment with etanercept. Etanercept treatment resulted in an improvement of symptoms due to RA and in a reduction of urinary excretion levels of PYD and DPD as well as serum sRANKL levels, with a significant difference at 6 months, and an increase of serum BAP levels at 3 and 6 months after the initial treatment with etanercept. Urinary NTX and serum OPG levels did not show a significant change at 3 and 6 months after the initial treatment, but serum OPG levels did show a reverse correlation with serum CRP levels, suggesting that the regulation of inflammation in RA may result in an induction of OPG production. Etanercept may have the ability to reduce the levels of bone resorption markers and to increase the levels of a bone formation marker while reducing sRANKL formation in RA patients.     

Evaluation of bone mineral density, bone metabolism, osteoprotegerin and receptor activator of the NFkappaB ligand serum levels during treatment with infliximab in patients with rheumatoid arthritis

OBJECTIVES: To examine whether treatment with anti-tumour necrosis factor (TNF) alpha prevents loss of bone mineral density (BMD) at the spine and hip (generalised) and in the hands (local) of patients with rheumatoid arthritis, and to study the changes in markers of bone metabolism, including receptor activator of the NFkappaB ligand (RANKL) and osteoprotegerin (OPG), during anti-TNF treatment. PATIENTS AND METHODS: 102 patients with active rheumatoid arthritis, who were treated with infliximab during 1 year, were included in this open cohort study. The BMD of the spine and hip (dual x ray absorptiometry) and hands dual x ray radiogrammetry was measured before the start of treatment and after 1 year. Changes in osteocalcin formation, beta-isomerised carboxy terminal telopeptide of type 1 collagen (beta-CTx, resorption), RANKL and OPG were determined at 0, 14, 30 and 46 weeks. RESULTS: The BMD of the spine and hip was unchanged during treatment with infliximab, whereas BMD of the hand decreased significantly by 0.8% (p<0.01). The BMD of the hip in patients with a good European League Against Rheumatism response showed a favourable change compared with patients not achieving such a response. Serum beta-CTx and RANKL were both considerably decreased compared with baseline at all time points. The decrease in beta-CTx was associated with the decrease in Disease Activity Score of 28 joints and C reactive protein during the 0-14 weeks interval. CONCLUSION: In patients with rheumatoid arthritis treated with infliximab, spine and hip bone loss is arrested, whereas metacarpal cortical hand bone loss is not stopped. The outcome of the study also supports a relationship between clinical response, in terms of reduced inflammatory activity, and changes in bone loss of the spine, hip and hands.     

Laboratory changes on anti-tumor necrosis factor treatment in rheumatoid arthritis

Tumor necrosis factor-alpha, acting through its receptors expressed on all cells of the body, is a key mediator of inflammation and immunity. However, its overproduction may also lead to pathologic changes. The latter situation occurs often in chronic inflammatory diseases such as rheumatoid arthritis. The concept suggesting tumor necrosis factor-alpha as a potential target emerged from experiments showing its key role in inducing many cytokines and mediators of inflammation. Several clinical trials targeting this cytokine in rheumatoid arthritis patients with a novel group of anti-tumor necrosis factor agents demonstrated reduced synovial inflammation and inhibition of bone and cartilage degradation. In addition to the therapeutic value of anti-tumor necrosis factor, analysis of laboratory changes not only proved the concept but provided new data, continuously expanding our understanding of the role of tumor necrosis factor-alpha in the pathogenesis of many diseases. These laboratory measures may also help the earlier identification of rheumatoid arthritis patients who have a less satisfactory response to this therapy.     

Osteoprotegerin and receptor activator of nuclear factor kappaB ligand expression in fibroblast-like synoviocytes from rheumatoid arthritis and osteoarthritis patients

SIR, We read with interest the article by Haynes et al. [1]reporting the expression of osteoprotegerin (OPG) and receptoractivator of nuclear factor B ligand (RANKL) in synovial tissuesfrom patients with arthritis, including rheumatoid arthritis(RA). They showed that OPG was expressed predominantly in macrophagesin the synovial lining layer and in endothelial cells, and expressionwas decreased in patients with active RA compared with thosewith osteoarthritis (OA), inactive RA and spondyloarthropathies.In contrast, RANKL expression was seen in active RA synovialtissues,