Osteoprotegerin expression in synovial tissue from patients with rheumatoid arthritis,spondyloarthropathies and osteoarthritis and normal controls

OBJECTIVES: To demonstrate the expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappaB ligand (RANKL) in synovial tissue from rheumatoid arthritis (RA) patients, establish the cell lineage expressing OPG and compare the expression of OPG in RA, spondyloarthropathies, osteoarthritis and normal synovial tissue. METHODS: Synovial biopsy specimens were obtained at arthroscopy from 16 RA and 12 spondyloarthropathy patients with active synovitis of a knee joint, six RA patients with no evidence of active synovitis, 10 patients with osteoarthritis and 18 normal subjects. Immunohistological analysis was performed using monoclonal antibodies (mAb) to detect OPG and RANKL expression. In addition, dual immunohistochemical evaluation was performed with lineage-specific monoclonal antibodies (macrophages, fibroblasts and endothelial cells) and OPG to determine the cell lineages expressing OPG. The sections were evaluated by computer-assisted image analysis and semiquantitative analysis. RESULTS: Two patterns of OPG expression were seen, one exclusively in endothelial cells and one expressed predominantly in macrophages in the synovial lining layer. Both patterns of OPG staining could be blocked with excess recombinant OPG. Endothelial and synovial lining expression of OPG was seen in all synovial tissues except those from patients with active RA. In contrast, RANKL expression was seen predominantly in synovial tissue from patients with active disease, mainly in sublining regions, particularly within areas of lymphocyte infiltration. CONCLUSIONS: OPG expression on macrophage type synovial lining cells as well as endothelial cells is deficient in RA patients with active synovitis, in contrast to that seen in spondyloarthropathy patients with active synovitis. This deficiency in OPG expression in the inflamed joint of RA patients may be important in the development of radiologically defined joint erosions.     

The abundant synovial expression of the RANK/RANKL/Osteoprotegerin system in peripheral spondylarthritis is partially disconnected from inflammation

OBJECTIVE: Spondylarthritis (SpA) and rheumatoid arthritis (RA) have different patterns of bone damage, with more pronounced bone erosions in RA. The RANK/RANKL/osteoprotegerin (OPG) system plays a central role in bone resorption by promoting the maturation and activation of osteoclasts. To assess the potential role of this system in the distinct bone phenotype, we studied the synovial expression of these mediators in SpA and RA peripheral synovitis. METHODS: Synovial biopsy specimens were obtained from the actively inflamed peripheral joints of 35 patients with SpA and 19 patients with RA. Paired synovial biopsy samples were obtained from 24 patients with SpA after tumor necrosis factor alpha (TNFalpha) blockade. Synovial tissue sections were immunostained for RANKL, OPG, RANK, and TRAP and assessed by semiquantitative scoring and digital image analysis. RESULTS: After extensive validation of the reactivity and specificity of the antibodies, we demonstrated the abundant expression of RANKL and OPG in SpA synovitis. RANKL was expressed by both fibroblast-like synoviocytes and sublining T lymphocytes. RANK-positive osteoclast precursors but no mature TRAP-positive osteoclasts were present in the inflamed tissue. The expression of these mediators was not different between patients with nonpsoriatic SpA, patients with psoriatic SpA, and patients with RA, was not related to the degree of systemic or local inflammation, and was not significantly modulated by highly effective treatment with TNFalpha blockers. Only the subset of patients with the best systemic response to TNFalpha blockade had decreased RANKL expression in the intimal lining layer. CONCLUSION: The relative protection against bone erosions in SpA cannot be explained by qualitative or quantitative differences in the synovial expression of RANKL, OPG, and RANK. The abundant expression of these factors in SpA peripheral synovitis is largely disconnected from systemic and local inflammation.     

Modulation of RANKL and osteoprotegerin expression in synovial tissue from patients with rheumatoid arthritis in response to disease-modifying antirheumatic drug treatment and correlation with radiologic outcome

OBJECTIVE: To demonstrate the effect of treatment with disease-modifying agents on the expression of osteoprotegerin (OPG) and RANKL in the synovial tissue from rheumatoid arthritis (RA) patients and to correlate these changes with radiologic damage measured on sequential radiographs of the hands and feet. METHODS: Synovial biopsy specimens were obtained at arthroscopy from 25 patients with active RA (16 of whom had a disease duration <12 months) before and at 3-6-month intervals after starting treatment with a disease-modifying agent. Immunohistologic analysis was performed using monoclonal antibodies to detect OPG and RANKL expression, with staining quantitated using computer-assisted image analysis and semiquantitative analysis techniques. Serial radiographs of the hands and feet were analyzed independently by 2 radiologists and a rheumatologist using the van der Heide modification of the Sharp scoring method. RESULTS: Thirteen patients achieved a low disease state as defined by a disease activity score <2.6 while 19 patients achieved an American College of Rheumatology response >20% after disease-modifying antirheumatic drug (DMARD) treatment. Successful DMARD treatment resulted in an increase in OPG expression and a decrease in RANKL expression at the synovial tissue level, which correlated with a reduction in erosion scores measured on annual radiographs of the hands and feet. CONCLUSION: Successful treatment-induced modulation of OPG and RANKL expression at the synovial tissue level, resulting in a reduction in the RANKL:OPG ratio, is likely to have a significant impact on osteoclast formation and joint damage in patients with active RA.     

Anti-tumor necrosis factor therapy increases synovial osteoprotegerin expression in rheumatoid arthritis

OBJECTIVE: Treatment of rheumatoid arthritis (RA) with tumor necrosis factor (TNF)-blocking agents, including etanercept and infliximab, has resulted in reductions in the radiographic progression of RA. However, the exact mechanism by which this protection occurs has not been determined. In order to add to such knowledge, we investigated the effect of anti-TNF therapy on the expression of osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL) in synovial tissue. METHODS: The expression of OPG and RANKL in synovial biopsy specimens was evaluated by immunohistochemistry. Serial synovial biopsy specimens were obtained from 18 patients with RA, before and after treatment with etanercept (9 patients) or infliximab (9 patients). Biopsy specimens were evaluated by double-blind semiquantitative analysis and image analysis. The in vitro effect of TNF antagonists on the RANKL/OPG expression in osteoblasts and endothelial cells was evaluated by Western blotting. Statistical analysis was performed using Wilcoxon's signed rank test, followed by the Bonferroni correction for multiple comparisons of paired samples. The results of in vitro experiments were evaluated by one-way analysis of variance, with Tukey's post hoc test. RESULTS: Treatment with both infliximab and etanercept increased the expression of OPG in synovial tissue. After 8 weeks of treatment, neither infliximab nor etanercept influenced RANKL expression. In both groups of patients, the RANKL:OPG ratio decreased following therapy. In vitro, both of the TNF antagonists mimicked the in vivo effect, inducing a decrease in the RANKL:OPG ratio in TNF-primed osteoblasts and endothelial cells. CONCLUSION: Therapy with TNF antagonists in RA modulates the OPG/RANKL system, a potential mechanism that could explain the retardation of radiographic damage observed following anti-TNF therapy.     

Receptor activator NF-kappaB ligand (RANKL) expression in synovial tissue from patients with rheumatoid arthritis,spondyloarthropathy, osteoarthritis,and from normal patients: semiquantitative and quantitative analysis

OBJECTIVES: To compare receptor activator of NF-kappaB ligand (RANKL) production in the synovial tissue from patients with active rheumatoid arthritis (RA), inactive RA, spondyloarthropathies (SpA), osteoarthritis, and from normal subjects. In addition, to establish the cell lineages expressing RANKL in these tissues. METHODS: Immunohistological analysis of frozen synovial tissue biopsy specimens was performed using a monoclonal antibody (mAb) to detect RANKL. Sections were evaluated by computer assisted image analysis and semiquantitative analysis to compare RANKL expression between groups. Dual and sequential labelling with mAb RANKL and cell lineage specific monoclonal antibodies were used to determine the types of cells expressing RANKL. RESULTS: Higher levels of RANKL were expressed in tissues from patients with active RA and SpA than in tissues from patients with inactive RA, osteoarthritis, and from normal subjects. RANKL protein was associated with CD3 antigen-positive lymphocytes and some macrophages. RANKL was predominantly associated with activated, memory T cells (CD45Ro positive cells) in patients with active RA and spondyloarthropathy (SpA). CONCLUSIONS: The highest levels of RANKL were detected in patients with RA with active synovitis and in some patients with SpA. An increase in RANKL in the inflamed joint of patients with RA, produced by infiltrating activated T cells and macrophages, is likely to be an important cause of joint erosions in RA.     

Anti–tumor necrosis factor therapy increases synovial osteoprotegerin expression in rheumatoid arthritis

Objective

Treatment of rheumatoid arthritis (RA) with tumor necrosis factor (TNF)–blocking agents, including etanercept and infliximab, has resulted in reductions in the radiographic progression of RA. However, the exact mechanism by which this protection occurs has not been determined. In order to add to such knowledge, we investigated the effect of anti‐TNF therapy on the expression of osteoprotegerin (OPG) and receptor activator of NF‐κB ligand (RANKL) in synovial tissue.

Methods

The expression of OPG and RANKL in synovial biopsy specimens was evaluated by immunohistochemistry. Serial synovial biopsy specimens were obtained from 18 patients with RA, before and after treatment with etanercept (9 patients) or infliximab (9 patients). Biopsy specimens were evaluated by double‐blind semiquantitative analysis and image analysis. The in vitro effect of TNF antagonists on the RANKL/OPG expression in osteoblasts and endothelial cells was evaluated by Western blotting. Statistical analysis was performed using Wilcoxon's signed rank test, followed by the Bonferroni correction for multiple comparisons of paired samples. The results of in vitro experiments were evaluated by one‐way analysis of variance, with Tukey's post hoc test.

Results

Treatment with both infliximab and etanercept increased the expression of OPG in synovial tissue. After 8 weeks of treatment, neither infliximab nor etanercept influenced RANKL expression. In both groups of patients, the RANKL:OPG ratio decreased following therapy. In vitro, both of the TNF antagonists mimicked the in vivo effect, inducing a decrease in the RANKL:OPG ratio in TNF‐primed osteoblasts and endothelial cells.

Conclusion

Therapy with TNF antagonists in RA modulates the OPG/RANKL system, a potential mechanism that could explain the retardation of radiographic damage observed following anti‐TNF therapy.

     

Imbalanced expression of RANKL and osteoprotegerin mRNA in pannus tissue of rheumatoid arthritis

OBJECTIVE: To test if the pannus tissue is characterized by a high receptor activator of nuclear factor kappaB ligand to osteoprotegerin (RANKL:OPG) ratio, which could explain local osteoclastogenesis and formation of bony erosions. METHODS: Messenger RNA and protein expressions of RANKL and OPG in rheumatoid and osteoarthritic tissue samples were measured using quantitative real-time RT-PCR and Western blot/densitometry. Pannus and synovitis fibroblasts explanted from tissue samples were cultured in vitro without and with TNF-alpha, IL-1Beta or IL-17 and analyzed quantitatively for RANKL expression. The ability of pannus fibroblasts to induce formation of multinuclear osteoclast-like cells from human monocytes, with macrophage-colony stimulating factor (M-CSF) but without RANKL added, was tested. Histochemical staining was used to assess the eventual presence of RANKL and tartrate resistant acid phosphatase positive osteoclast-like cells at the pannus-bone interface. RESULTS: RANKL:OPG ratios of messenger RNA (p<0.05) and protein level were high in pannus (2.06+/-0.73 and 2.2+/-0.65) compared to rheumatoid (0.62+/-0.13 and 1.31+/-0.69) and osteoarthritis (0.62+/-0.32 and 0.52+/-0.16) synovial membranes. Resting and stimulated (p dependent on the cytokine used) pannus fibroblasts produced RANKL in excess (p=0.0005) and unstimulated pannus fibroblasts also effectively induced osteoclast-like cell formation from monocytes in vitro without any exogenous RANKL added. Compatible with these findings, multinuclear osteoclasts-like cells were frequent in the fibroblast- and macrophage-rich pannus tissue at the soft tissue-to-bone interface. CONCLUSION: The high RANKL:OPG ratio, together with close fibroblast-to-monocyte contacts in pannus tissue, probably favor local generation of bone resorbing osteoclasts at the site of erosion in rheumatoid arthritis.     

Activated human T cells directly induce osteoclastogenesis from human monocytes: possible role of T cells in bone destruction in rheumatoid arthritis patients

OBJECTIVE: To elucidate the direct role of human T cells in the induction of osteoclastogenesis in rheumatoid arthritis (RA), by studying human monocytes and the pathogenetic roles of receptor activator of nuclear factor kappaB ligand (RANKL), RANK, and osteoprotegerin (OPG). METHODS: Synovial tissue obtained at total knee replacement was stained immunohistologically using anti-RANKL, CD3, and CD4 antibodies. Synovial fluid was obtained from patients with RA, osteoarthritis (OA), gout, or trauma. Concentrations of the soluble form of RANKL (sRANKL) and OPG in the synovial fluid were measured by enzyme-linked immunosorbent assay. Activated T cells from peripheral blood mononuclear cells (PBMC) of healthy volunteers were cultured with human monocytes from PBMC. RESULTS: Immunostaining of the synovial tissue of RA patients demonstrated that RANKL-positive cells were detected in a subset of fibroblast-like synoviocytes and infiltrating mononuclear cells. Double immunostaining revealed that RANKL-positive cells were detected in a subset of CD3+ cells and CD4+ cells. An increased concentration of sRANKL and a decreased concentration of OPG were detected in synovial fluid from RA patients. The ratio of the concentration of sRANKL to that of OPG was significantly higher in synovial fluid of RA patients than in synovial fluid of patients with OA or gout. The activated T cells expressing RANKL induced osteoclastogenesis from autologous peripheral monocytes. The role of RANKL in this osteoclastogenetic process was confirmed by dose-dependent inhibition by OPG. CONCLUSION: The present study is the first to demonstrate osteoclastogenesis using human-derived T cells and monocytes. In addition, the present findings suggest that excess production of RANKL by activated T cells increases the level of sRANKL in synovial fluid and may contribute to osteoclastic bone resorption in RA patients.     

Histological features of rheumatoid arthritis patients having antibodies to enterobacterial common antigens: correlation of antibody levels with semiquantitative histologic scores and laboratory markers

OBJECTIVE: To ascertain whether clinically diagnosed rheumatoid arthritis (RA) patients having antibodies to enterobacterial common antigens (ECA) in synovial fluid (SF) have a histological appearance characteristic of RA synovitis. METHODS: Twenty-five RA patients for which synovial biopsy specimens were preserved, were selected from 58 patients with RA tested for antibodies to ECA in SF The synovial tissue specimens were examined histologically using a semiquantitative scoring system with quantitative counts. The correlation of anti-ECA antibody levels with total scores for synovitis and laboratory markers in three RA patient groups (markedly positive, moderately to slightly positive, and negative according to anti-ECA antibody levels) was analyzed statistically. RESULTS: Histologic examination in the markedly positive RA group (total score for synovitis, range 18-20 points) revealed typical histological features of rheumatoid synovitis. Total scores for synovitis were significantly higher in both the markedly positive and moderately to slightly positive RA groups than in the negative RA group. A comparison of anti-ECA antibody levels with total scores for synovitis revealed a strong and significant correlation. Furthermore, levels of anti-ECA antibodies were also correlated with rheumatoid factor and C-reactive protein. CONCLUSION: Clinically diagnosed RA patients having anti-ECA antibodies in SF showed typical or characteristic histological features of RA synovitis. Our data suggest that a group of RA patients with an entrobacterial etiology exists in larger groups of patients with RA which is thought to be a heterogeneous disease.     

Intraarticular corticosteroids decrease synovial RANKL expression in inflammatory arthritis

OBJECTIVE: Intraarticular corticosteroids are frequently used as successful adjuvant therapy for inflammatory arthritides, but little is known about their effects on molecules that regulate bone biology. We undertook this study to investigate the effect of intraarticular corticosteroids on the synovial expression of RANKL and osteoprotegerin (OPG). METHODS: We evaluated RANKL, OPG, and surface marker expression by immunohistochemical methods in synovial knee biopsy samples obtained from 13 patients with inflammatory arthritis before and 2 weeks following intraarticular injection of triamcinolone hexacetonide. We further investigated the effect of dexamethasone (DEX) on RANKL expression by lymphocytes from rheumatoid arthritis synovial fluids (RA SF), using flow cytometric analysis. Finally, we evaluated the in vitro effect of DEX on RANKL and OPG expression in osteoblast-like cells, by Western blotting. RESULTS: Intraarticular corticosteroids induced a decrease in the number of synovial T cells without influencing the number of macrophages, evaluated as both CD68+ and CD163+ cells. This change was paralleled by a decrease of synovial RANKL expression with a concomitant reduction of the RANKL:OPG ratio. DEX down-regulated RANKL expression on lymphocytes derived from RA SF. Moreover, in vitro pretreatment of osteoblast-like cells with tumor necrosis factor favored an antiresorptive effect of DEX treatment through a similar down-regulation of RANKL expression. CONCLUSION: The decrease in inflammation attributed to intraarticular corticosteroids is accompanied by down-modulation of bone destruction markers. These findings offer a rationale for the beneficial effect of corticosteroids on joint erosion in arthritis.     

Synovial tissue rank ligand expression and radiographic progression in rheumatoid arthritis: observations from a proof-of-concept randomized clinical trial of cytokine blockade

The objective of the study was to evaluate synovial tissue receptor activator of nuclear factor-κβ ligand (RANKL) and osteoprotegerin (OPG) as biomarkers of disease activity, progressive joint damage, and therapeutic response, during cytokine blockade in rheumatoid arthritis (RA). Patients with active RA entered a randomized open-label 12-month study of anakinra 100 mg/day, administered as monotherapy or in combination with pegsunercept 800 μg/kg twice weekly. Arthroscopic synovial tissue biopsies were obtained at baseline, at 4 weeks and at the final time point. Following immunohistochemical staining, RANKL and OPG expression was quantified using digital image analysis. Radiographic damage was evaluated using the van der Heijde modification of the Sharp scoring system. Twenty-two patients were randomized. Baseline expression of RANKL, but not OPG, correlated significantly with baseline CRP levels (r = 0.61, P < 0.01). While a significant reduction in OPG expression following treatment was observed in clinical responders at the final time point (P < 0.05 vs. baseline), RANKL levels did not change, and the RANKL:OPG ratio remained unaltered, even at the highest levels of clinical response. When potential predictors of radiographic outcome were evaluated, baseline RANKL expression correlated with erosive progression at 1 year (r = 0.71, P < 0.01). Distinct, though related, pathophysiologic processes mediate joint inflammation and destruction in RA. Elevated synovial tissue RANKL expression is associated with progressive joint erosion, and may be independent of the clinical response to targeted therapy. The potential therapeutic importance of modulating RANKL in RA is highlighted, if radiographic arrest is to be achieved.     

Involvement of receptor activator of nuclear factor kappaB ligand/osteoclast differentiation factor in osteoclastogenesis from synoviocytes in rheumatoid arthritis

OBJECTIVE: To clarify the mechanism by which osteoclasts are formed in culture of rheumatoid synoviocytes by exploring the involvement of receptor activator of nuclear factor kappaB ligand (RANKL)/osteoclast differentiation factor (ODF). METHODS: Osteoclast formation was evaluated in cocultures of rheumatoid synovial fibroblasts and peripheral blood mononuclear cells (PBMC) in the presence of macrophage colony stimulating factor and 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) utilizing separating membrane filters. RANKL/ODF expression was examined by Northern blotting in synovial tissues from 5 rheumatoid arthritis (RA) patients and tissues from patients with giant cell tumor (GCT), osteosarcoma (OS), and osteoarthritis (OA). RANKL/ODF expression and the ability of synovial fibroblasts to support osteoclastogenesis were investigated in coculture with PBMC in the presence or absence of 1,25(OH)2D3, and soluble RANKL/ODF and osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) were measured by enzyme-linked immunosorbent assay. The effects of OPG/OCIF on the osteoclastogenesis in the primary culture of rheumatoid synoviocytes and the coculture system were determined. RESULTS: Synovial fibroblasts did not induce osteoclastogenesis when separately cocultured with PBMC. Northern blotting revealed that RANKL/ODF was highly expressed in all tissues from RA and GCT patients, but not from OA or OS patients. Cultured rheumatoid synovial fibroblasts efficiently induced osteoclastogenesis in the presence of 1,25(OH)2D3, which was accompanied by up-regulated expression of RANKL/ODF and decreased production of OPG/OCIF. Osteoclastogenesis from synoviocytes was dose-dependently inhibited by OPG/OCIF. CONCLUSION: RANKL/ODF expressed on synovial fibroblasts is involved in rheumatoid bone destruction by inducing osteoclastogenesis and would therefore be a good therapeutic target.     

Cytokine-controlled RANKL and osteoprotegerin expression by human and mouse synovial fibroblasts: fibroblast-mediated pathologic bone resorption

OBJECTIVE: To determine whether proinflammatory cytokine treatment or the complete absence of select cytokines modulates the expression of RANKL and osteoprotegerin (OPG) in synovial fibroblasts. METHODS: Fibroblasts were isolated from normal and rheumatoid human synovium and from normal or arthritic joints of wild-type and cytokine gene-deficient (interleukin-4-knockout [IL-4 (-/-)] and interferon-gamma-knockout [IFNgamma (-/-)]) mice. Fibroblasts were stimulated with proinflammatory cytokines (tumor necrosis factor alpha [TNFalpha], IL-1beta, and IL-17) or antiosteoclastogenic cytokines (IL-4 and IFNgamma), alone or in combination, and the expression of RANKL and OPG was measured. RESULTS: Proinflammatory cytokine-stimulated fibroblasts from rheumatoid and arthritic mouse joints expressed higher levels of RANKL and OPG than those from normal joints. IL-4 suppressed RANKL expression and increased OPG expression, IFNgamma reduced the production of both RANKL and OPG, and IL-17 had only a modest effect on the expression of RANKL or OPG. Additive effects of combination treatment (TNFalpha/IL-17 or IL-1beta/IL-17) were observed only in the human system. Extensive destruction was observed in the arthritic joints of IL-4 (-/-) mice, with a corresponding upward shift of the RANKL:OPG ratios. However, an IL-17 deficiency did not attenuate arthritis or reduce bone resorption. CONCLUSION: Proinflammatory cytokines induce the expression of RANKL and OPG in both human and murine synovial fibroblasts. The RANKL:OPG ratios are shifted in favor of bone protection by IL-4 treatment, and, to a lesser extent, by IFNgamma treatment. Unexpectedly, an IL-17 deficiency alone does not induce reduced inflammatory bone destruction. Our results suggest that synovial fibroblasts may significantly contribute to bone resorption through modulation of RANKL and OPG production in a cytokine-rich milieu of inflamed joints.     

Abundant expression of common cytokine receptor gamma chain (CD132) in rheumatoid joints

OBJECTIVE: Activated macrophages upregulate surface expression of common cytokine receptor gamma chains (gammac, CD132), triggering of which induces secretion of proinflammatory cytokines. Rheumatoid synovial tissues contain a number of macrophages that play a pathogenic role by secreting proinflammatory cytokines. We studied the expression of gammac in the rheumatoid synovial tissues. METHODS: Cryosections of synovial tissues from patients with active rheumatoid arthritis (RA) or with osteoarthritis (OA) were stained with an anti-gammac Mab. Single-cell suspensions from the rheumatoid synovial tissues were stained with the same antibody and an anti-CD14 monoclonal antibody (Mab) for 2-color flow cytometric analysis. A soluble form of gammac in synovial fluids collected from rheumatoid or OA joints was quantitated by ELISA. RESULTS: Rheumatoid synovial tissues, but not the OA tissues, expressed gammac at a high level. Flow cytometric analysis showed that gammac were expressed by virtually all CD 14 positive synovial cells in RA. Synovial fluid derived from the rheumatoid joints contained a high concentration of soluble gammac. CONCLUSION: Membrane bound and soluble forms of gammac are abundant in rheumatoid joints. They might play a complex role in the pathology of RA.     

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,     

Detection of cytokine producing cells in the synovial membrane from patients with rheumatoid arthritis.

OBJECTIVES--To develop and evaluate a new immunohistochemical method to study the localisation and phenotype of individual cytokine producing cells in synovial biopsy specimens in rheumatoid arthritis. METHODS--Cryopreserved sections of synovial tissue from nine patients with rheumatoid arthritis were incubated with carefully selected cytokine specific antibodies detecting 19 different cytokines, after fixation of the specimens with paraformaldehyde and using saponin to permeabilise the cell membranes. RESULTS--The immunohistochemical method yielded reproducible and distinct staining patterns, in which the cytokines accumulated mainly in the Golgi apparatus of producer cells, indicating that the method preferentially detected local synthesis rather than cytokine uptake. The cytokine production patterns varied considerably between biopsy specimens from different patients. CONCLUSION--The present modified immunohistochemical method may provide a simple and rapid way to determine the local production of a wide array of cytokines in the synovium. The data obtained with this method also indicated that more T cell derived cytokines than previously recognised were present in active synovitis, as located and sampled by arthroscopy.     

Absence of histologic evidence of synovitis in patients with Gulf War veterans' illness with joint pain

OBJECTIVE: An unexplained multisymptom illness, Gulf War veterans' illness (GWVI), has been described among allied force veterans of the first Gulf War (1990-1991). It has been proposed that some of its symptoms reflect an immune dysfunction, and rheumatologic symptoms including joint pain and stiffness are reported frequently. However, it is unknown whether synovial inflammation causes the articular symptoms. We examined synovial tissue from individuals with GWVI and joint pain for evidence of inflammation. METHODS: We compared synovial biopsy samples from 6 individuals with GWVI and joint pain with samples from 9 clinically asymptomatic controls (hematoxylin and eosin [H&E] stains only) and biopsy samples or surgically obtained specimens from 10 patients with rheumatoid arthritis (RA) and 12 with osteoarthritis (OA). Inflammatory changes were quantified in H&E stained sections with a modified synovitis score by immunostaining for CD3, CD20, CD38, CD68, Ki-67, and von Willebrand factor, and with a composite inflammation score based on these markers. RESULTS: Normal histology was seen in the GWVI specimens, except for mild focal lining hyperplasia and rare low-grade perivascular infiltrates in 1 specimen each. Mean +/- SEM synovitis scores were lowest and nearly identical in control (1.38 +/- 0.30) and GWVI specimens (1.41 +/- 0.29), intermediate in OA specimens (2.64 +/- 0.39), and highest in RA specimens (6.0 +/- 0.19). Likewise, inflammatory cells, cell division, vascular density, and composite inflammation score were lowest in the GWVI specimens. CONCLUSION: Despite significant joint pain, the GWVI synovia did not differ from normal controls. These results agree with other studies that have failed to document inflammatory or immunologic etiologies in GWVI.     

Inflammatory cell infiltrate and RANKL/OPG expression in rheumatoid synovium: comparison with other inflammatory arthropathies and correlation with outcome

OBJECTIVES: To evaluate if the immunofluorescence analysis of synovial tissue (ST) using antibodies against RANKL/OPG, conjugated with the immunophenotyping of lymphocytes and macrophages, could be of diagnostic and prognostic value in rheumatoid arthritis (RA) patients. METHODS: 3-year prospective study of 103 consecutive patients submitted to closed needle biopsy for diagnostic purposes. ST was analyzed with routine histologic techniques and immunofluorescence, using monoclonal antibodies against RANKL, OPG, CD163, CD68, CD4, CD8, interferon-gamma and CD19. Patients were prospectively evaluated with a clinical, laboratorial and radiological protocol. At the end of the follow-up patients were divided according to the final diagnosis. Results of the initial histologic evaluation were compared between the main diagnostic groups and in RA patients histologic data was correlated with clinical and radiologic outcome measures. RESULTS: The RANKL/OPG ratio and the inflammatory infiltrate were significatively higher in RA (n = 25) as compared to the same ratio observed in other inflammatory joint diseases (OIJD, n = 48) and in osteoarthritis (n = 17). The difference between RA and OIJD was specifically confirmed when the comparison involved spondyloarthropathy (n = 26). Final HAQ score and radiologic outcome were correlated with the density of intimal CD68+ macrophages. Radiologic progression was correlated with subintimal CD4+ lymphocytes and CD68+ macrophages and intimal CD68 and CD163+ macrophages. CONCLUSION: The quantification of the RANKL/OPG ratio and of the number of lymphocytes in the ST might be useful to differentiate RA from other inflammatory joint diseases. The ST number of CD4+ lymphocytes and macrophages are probable predictors of radiologic progression in RA patients.     

IMMUNOHISTOLOGICAL FEATURES IN THE SYNOVIUM OBTAINED FROM CLINICALLY UNINVOLVED KNEE JOINTS OF PATIENTS WITH RHEUMATOID ARTHRITIS

The spectrum of immunohistological change in the affected jointsof patients with rheumatoid arthritis has been well described.In this study, the immunohistological features in synovial membraneobtained from apparently uninvolved knee joints of 16 patientswith active untreated rheumatoid arthritis were examined andcompared to tissue from control subjects. Synovial tissue wasobtained by needle biopsy. Hyperplasia of the synovial lininglayer, present in 69%, was the most frequently observed abnormalityin synovium obtained from uninvolved joints. Perivascular mononuclearcell infiltration was present in 31% and consisted predominantlyof helper T-cells. Increased vascularity and fibrin depositionwere not notable features. Clinically overt synovitis emergedin only two patients during a follow-up period of up to 36 months.In conclusion, a considerable degree of histological changewas observed in the apparently uninvolved knee joints of patientswith active rheumatoid arthritis. The presence of subclinicalsynovitis challenges current concepts of disease activity andclinical remission. Further study is required to determine whetherthe features described may be associated with progressive jointerosion. KEY WORDS: Synovial biopsy, Inflammation, Synovitis, Suppressor/cytotoxic T-cells, Helper T-cells, B-cells     

Expression of interferon beta in synovial tissue from patients with rheumatoid arthritis: comparison with patients with osteoarthritis and reactive arthritis

BACKGROUND: IFNbeta may have immunomodulatory effects in rheumatoid arthritis (RA) and its increased production in RA synovium may be a reactive attempt to inhibit inflammation. OBJECTIVE: To determine the expression of IFNbeta in the synovial tissue of patients with RA, osteoarthritis, and reactive arthritis. METHODS: Synovial biopsy specimens were obtained by arthroscopy from patients with RA and disease controls for immunohistological analysis using a monoclonal antibody specific for IFNbeta. Bound antibody was detected by an immunoperoxidase method. Stained sections were evaluated by computer assisted image analysis. Double stainings were performed with antibodies to detect CD55 positive fibroblast-like synoviocytes (FLS), CD68 positive macrophages, and CD83 positive dendritic cells (DCs) co-expressing IFNbeta. RESULTS: IFNbeta protein was abundantly expressed in the synovium of patients with RA. Digital image analysis showed a significant increase in the mean integrated optical density for IFNbeta expression in RA synovial tissue compared with disease controls. Specific up regulation of IFNbeta expression was also seen when the results were controlled for cell numbers. Phenotypic analysis showed that FLS, especially, but also macrophages and DCs may express IFNbeta in RA synovial tissue. CONCLUSIONS: The increased expression of IFNbeta in RA synovium suggests activation of an immunomodulatory mechanism that could inhibit synovial inflammation.