Dynamic contrast-enhanced magnetic resonance imaging of metacarpophalangeal joints reflects histological signs of synovitis in rheumatoid arthritis

Introduction

Synovial inflammation and joint destruction in rheumatoid arthritis (RA) may progress despite clinical remission. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is increasingly used to detect synovial inflammation in RA. Although small joints such as metacarpophalangeal (MCP) joints are mainly affected by RA, MRI findings have never been directly compared to histological synovitis in MCP synovial tissue. The objective of the current study was therefore to analyse if DCE-MRI relates to histological signs of synovitis small RA joints.

Methods

In 9 RA patients, DCE-MRI (3 Tesla, dynamic 2D T1 weighted turbo-flash sequence) of the hand was performed prior to arthroscopically-guided synovial biopsies from the second MCP of the imaged hand. Maximum enhancement (ME), rate of early enhancement, and maximum rate of enhancement were assessed in the MCP. Synovial biopsies were stained for determination of sublining CD68 and the Synovitis Score. Correlations between MRI and histological data were calculated according to Spearman.

Results

ME of the MCP significantly correlated to sublining CD68 staining (r = 0.750, P = 0.02), the Synovitis Score (r = 0.743, P = 0.02), and the subscores for lining layer hypertrophy (r = 0.789, P = 0.01) and cellular density (r = 0.842; P = 0.004).

Conclusions

Perfusion imaging of synovial tissue in RA finger joints employing DCE-MRI reflects histological synovial inflammation. According to our study, ME is the most closely associated parameter amongst the measures considered.     

Synovial explant inflammatory mediator production corresponds to rheumatoid arthritis imaging hallmarks: a cross-sectional study

Introduction

Despite the widespread use of magnetic resonance imaging (MRI) and Doppler ultrasound for the detection of rheumatoid arthritis (RA) disease activity, little is known regarding the association of imaging-detected activity and synovial pathology. The purpose of this study was to compare site-specific release of inflammatory mediators and evaluate the corresponding anatomical sites by examining colour Doppler ultrasound (CDUS) and MRI scans.

Methods

RA patients were evaluated on the basis of CDUS and 3-T MRI scans and subsequently underwent synovectomy using a needle arthroscopic procedure of the hand joints. The synovial tissue specimens were incubated for 72 hours, and spontaneous release of monocyte chemoattractant protein 1 (MCP-1), interleukin 6 (IL-6), macrophage inflammatory protein 1β (MIP-1β) and IL-8 was measured by performing multiplex immunoassays. Bone marrow oedema (BME), synovitis and erosion scores were estimated on the basis of the rheumatoid arthritis magnetic resonance imaging score (RAMRIS). Mixed models were used for the statistical analyses. Parsimony was achieved by omitting covariates with P > 0.1 from the statistical model.

Results

Tissue samples from 58 synovial sites were obtained from 25 patients. MCP-1 was associated with CDUS activity (P = 0.009, approximate Spearman’s ρ = 0.41), RAMRIS BME score (P = 0.01, approximate Spearman’s ρ = 0.42) and RAMRIS erosion score (P = 0.03, approximate Spearman’s ρ = 0.31). IL-6 was associated with RAMRIS synovitis score (P = 0.04, approximate Spearman’s ρ = 0.50), BME score (P = 0.04, approximate Spearman’s ρ = 0.31) and RAMRIS erosion score (P = 0.03, approximate Spearman’s ρ = 0.35). MIP-1β was associated with CDUS activity (P = 0.02, approximate Spearman’s ρ = 0.38) and RAMRIS synovitis scores (P = 0.02, approximate Spearman’s ρ = 0.63). IL-8 associations with imaging outcome measures did not reach statistical significance.

Conclusions

The association between imaging activity and synovial inflammatory mediators underscores the high sensitivity of CDUS and MRI in the evaluation of RA disease activity. The associations found in our present study have different implications for synovial mediator releases and corresponding imaging signs. For example, MCP-1 and IL-6 were associated with both general inflammation and bone destruction, in contrast to MIP-1β, which was involved solely in general synovitis. The lack of association of IL-8 with synovitis was likely underestimated because of a large proportion of samples above assay detection limits among the patients with the highest synovitis scores.     

Outcome predictors of intra-articular glucocorticoid treatment for knee synovitis in patients with rheumatoid arthritis – a prospective cohort study

Introduction

Intra-articular glucocorticoid treatment (IAGC) is widely used for symptom relief in arthritis. However, knowledge of factors predicting treatment outcome is limited. The aim of the present study was to identify response predictors of IAGC for knee synovitis in patients with rheumatoid arthritis (RA).

Methods

In this study 121 RA patients with synovitis of the knee were treated with intra-articular injections of 20 mg triamcinolone hexacetonide. They were followed for six months and the rate of clinical relapse was studied. Non-responders (relapse within 6 months) and responders were compared regarding patient characteristics and knee joint damage as determined by the Larsen-Dale index. In addition, matched samples of serum and synovial fluid were analysed for factors reflecting the inflammatory process (C-reactive protein, interleukin 6, tumour necrosis factor alpha, vascular endothelial growth factor), joint tissue turnover (cartilage oligomeric matrix protein, metalloproteinase 3), and autoimmunity (antinuclear antibodies, antibodies against citrullinated peptides, rheumatoid factor).

Results

During the observation period, 48 knees relapsed (40%). Non-responders had more radiographic joint damage than responders (P = 0.002) and the pre-treatment vascular endothelial growth factor (VEGF) level in synovial fluid was significantly higher in non-responders (P = 0.002).

Conclusions

Joint destruction is associated with poor outcome of IAGC for knee synovitis in RA. In addition, higher levels of VEGF in synovial fluid are found in non-responders, suggesting that locally produced VEGF is a biomarker for recurrence of synovial hyperplasia and the risk for arthritis relapse.     

Anti-cyclic citrullinated peptide antibodies in primary Sjögren syndrome may be associated with non-erosive synovitis

Introduction

The purpose of this study was to investigate the prevalence of cyclic citrullinated peptide antibodies (anti-CCP) in patients with primary Sjögren syndrome (pSS) and its correlation with clinical and laboratory data.

Methods

We analysed the clinical and serological data of 155 consecutive patients with pSS. Among these, 14 were excluded due to fulfillment of American College of Rheumatology criteria for rheumatoid arthritis (RA). So, 141 patients (27 males and 114 females; mean age 48 years, range 39 to 60) were clinically assessed for the presence of synovitis (objective swelling of one or more joints) and extra-glandular involvement. The anti-CCP antibodies were tested using a commercially available second-generation enzyme-linked immunosorbent assay. IgM rheumatoid factor (RF) was determined by nephelometry.

Results

Fourteen patients (9.9%) had moderate to high levels of anti-CCP, and 94 (66.7%) were positive for RF. Eighty-one (57.4%) showed extra-glandular involvement, and 44 (31.2%) had synovitis without any radiographic sign of erosion. There was a close correlation between the presence of anti-CCP and synovitis (P < 0.001) but no association between anti-CCP and extra-glandular involvement (P = 0.77). Multivariate analysis confirmed the association between anti-CCP and an increased prevalence of synovitis (prevalence odds ratio for positive versus negative anti-CCP status 7.611, 95% confidence interval 1.475 to 74.870; P = 0.010).

Conclusion

Only a minority of patients with pSS are anti-CCP-positive, which seems to be closely associated with the prevalence of synovitis. Anti-CCP positivity in patients with pSS therefore may be a predictor of future progress to RA or an expression of the inflammatory process of synovial tissue.     

Linking Power Doppler Ultrasound to the Presence of Th17 Cells in the Rheumatoid Arthritis Joint

Background

Power Doppler ultrasound (PDUS) is increasingly used to assess synovitis in Rheumatoid Arthritis (RA). Prior studies have shown correlations between PDUS scores and vessel counts, but relationships with T cell immunopathology have not been described.

Methodology/Principal Findings

PBMC were isolated from healthy controls (HC) or RA patients and stimulated ex vivo with PMA and ionomycin for 3 hours in the presence of Golgistop. Paired synovial fluid (SF) or synovial tissue (ST) were analysed where available. Intracellular expression of IL-17, IFNγ, and TNFα by CD4+ T cells was determined by flow cytometry. Synovial blood flow was evaluated by PDUS signal at the knees, wrists and metacarpophalangeal joints of RA patients. Serum, SF and fibroblast culture supernatant levels of vascular endothelial growth factor-A (VEGF-A) were measured by ELISA. The frequency of IL17+IFNγ-CD4+ T cells (Th17 cells) was significantly elevated in peripheral blood (PB) from RA patients vs. HC (median (IQR) 0.5 (0.28–1.59)% vs. 0.32 (0.21–0.54)%, p = 0.005). Th17 cells were further enriched (mean 6.6-fold increase) in RA SF relative to RA PB. Patients with active disease had a higher percentage of IL-17+ T cells in ST than patients in remission, suggesting a possible role for Th17 cells in active synovitis in RA. Indeed, the percentage of Th17 cells, but not Th1, in SF positively correlated with CRP (r = 0.51, p = 0.04) and local PDUS-defined synovitis (r = 0.61, p = 0.002). Furthermore, patients with high levels of IL-17+CD4+ T cells in SF had increased levels of the angiogenic factor VEGF-A in SF. Finally, IL-17, but not IFNγ, increased VEGF-A production by RA synovial fibroblasts in vitro.

Conclusions/Significance

Our data demonstrate a link between the presence of pro-inflammatory Th17 cells in SF and local PDUS scores, and offer a novel immunological explanation for the observation that rapid joint damage progression occurs in patients with persistent positive PDUS signal.     

Patients with rheumatoid arthritis in clinical remission and ultrasound-defined active synovitis exhibit higher disease activity and increased serum levels of angiogenic biomarkers

Introduction

The aim of this study was to identify and characterize subclinical synovitis in patients with rheumatoid arthritis (RA) in clinical remission using power Doppler ultrasound (PDUS) and serum levels of biomarkers of inflammation and/or angiogenesis.

Methods

We selected patients with RA in clinical remission defined as a Disease activity score of 28 joints (DAS28)-erythrocyte sedimentation rate (ESR) <2.6 for more than six months tested by two independent rheumatologists. Clinical, epidemiological, demographic and serological data were analyzed. PDUS of knees and hands was performed by a sonographer. Synovial hypertrophy (SH) and PDUS signal were scored (grades 0 to 3). SH ≥2 and a PDUS signal was classified as active synovitis. Serum levels of biomarkers of inflammation/angiogenesis were determined by Quantibody^® Human Array.

Results

This study included 55 patients, of whom 25 (45.4%) met criteria for ultrasound-defined active synovitis. Patients with active synovitis had higher DAS28-C reactive protein (P = 0.023), DAS28-ESR (P = 0.06), simplified disease activity score, SDAI (P = 0.064), and only 12% were taking oral glucocorticoids (≤5 mg/day) compared with 40% of patients without active synovitis (P = 0.044). Patients with synovitis also had significantly higher serum levels of the angiogenic biomarkers angiopoietin-2 (P = 0.038), vascular endothelial growth factor-D (P = 0.018), placental growth factor (P = 0.043), stromal cell-derived factor-1 (P = 0.035), matrix metallopeptidase-2 (P = 0.027) and basic fibroblast growth factor (bFGF) (P = 0.007), but not of pro-inflammatory cytokines.In the multivariate logistic regression model used to explore prognostic biomarkers for active synovitis, serum levels of bFGF, DAS28-ESR and not receiving glucocorticoids were the best predictors of active synovitis. The predictive indexes provided by the model were specificity 73.3%, sensitivity 72%, and area under the curve in receiver operating characteristic 81.5% (95% CI: 70.1% to 92.8%).

Conclusions

Nearly half of the patients with RA in clinical remission had ultrasound-defined active synovitis, higher disease activity and less frequent oral glucocorticoid consumption than patients without active synovitis. This clinical situation was associated with a specific biological profile characterized by an excess of angiogenic mediators rather than persistent proinflammatory cytokine responses.     

The role of total and cartilage-specific estrogen receptor alpha expression for the ameliorating effect of estrogen treatment on arthritis

Introduction

Estrogen (E2) delays onset and decreases severity of experimental arthritis. The aim of this study was to investigate the importance of total estrogen receptor alpha (ERα) expression and cartilage-specific ERα expression in genetically modified mice for the ameliorating effect of estrogen treatment in experimental arthritis.

Methods

Mice with total (total ERα^-/-) or cartilage-specific (Col2α1-ERα^-/-) inactivation of ERα and wild-type (WT) littermates were ovariectomized, treated with E2 or placebo, and induced with antigen-induced arthritis (AIA). At termination, knees were collected for histology, synovial and splenic cells were investigated by using flow cytometry, and splenic cells were subjected to a T-cell proliferation assay.

Results

E2 decreased synovitis and joint destruction in WT mice. Amelioration of arthritis was associated with decreased frequencies of inflammatory cells in synovial tissue and decreased splenic T-cell proliferation. E2 did not affect synovitis or joint destruction in total ERα^-/- mice. In Col2α1-ERα^-/- mice, E2 protected against joint destruction to a similar extent as in WT mice. In contrast, E2 did not significantly ameliorate synovitis in Col2α1-ERα^-/- mice.

Conclusions

Treatment with E2 ameliorates both synovitis and joint destruction in ovariectomized mice with AIA via ERα. This decreased severity in arthritis is associated with decreased synovial inflammatory cell frequencies and reduced splenic T-cell proliferation. ERα expression in cartilage is not required for estrogenic amelioration of joint destruction. However, our data indicate that ERα expression in cartilage is involved in estrogenic effects on synovitis, suggesting different mechanisms for the amelioration of joint destruction and synovitis by E2.     

Increased physical activity severely induces osteoarthritic changes in knee joints with papain induced sulfate-glycosaminoglycan depleted cartilage

Introduction

Articular cartilage needs sulfated-glycosaminoglycans (sGAGs) to withstand high pressures while mechanically loaded. Chondrocyte sGAG synthesis is regulated by exposure to compressive forces. Moderate physical exercise is known to improve cartilage sGAG content and might protect against osteoarthritis (OA). This study investigated whether rat knee joints with sGAG depleted articular cartilage through papain injections might benefit from moderate exercise, or whether this increases the susceptibility for cartilage degeneration.

Methods

sGAGs were depleted from cartilage through intraarticular papain injections in the left knee joints of 40 Wistar rats; their contralateral joints served as healthy controls. Of the 40 rats included in the study, 20 rats remained sedentary, and the other 20 were subjected to a moderately intense running protocol. Animals were longitudinally monitored for 12 weeks with in vivo micro-computed tomography (μCT) to measure subchondral bone changes and single-photon emission computed tomography (SPECT)/CT to determine synovial macrophage activation. Articular cartilage was analyzed at 6 and 12 weeks with ex vivo contrast-enhanced μCT and histology to measure sGAG content and cartilage thickness.

Results

All outcome measures were unaffected by moderate exercise in healthy control joints of running animals compared with healthy control joints of sedentary animals. Papain injections in sedentary animals resulted in severe sGAG-depleted cartilage, slight loss of subchondral cortical bone, increased macrophage activation, and osteophyte formation. In running animals, papain-induced sGAG-depleted cartilage showed increased cartilage matrix degradation, sclerotic bone formation, increased macrophage activation, and more osteophyte formation.

Conclusions

Moderate exercise enhanced OA progression in papain-injected joints and did not protect against development of the disease. This was not restricted to more-extensive cartilage damage, but also resulted in pronounced subchondral sclerosis, synovial macrophage activation, and osteophyte formation.     

Quantitative assessment of synovitis in patients with rheumatoid arthritis using fluorescence optical imaging

Introduction

To prospectively evaluate quantitative assessment of fluorescence optical imaging (FOI) for differentiation of synovitic from non-synovitic joints in patients suffering from rheumatoid arthritis (RA).

Methods

FOI of the hands was performed in patients with active RA, and a stratified quantitative fluorescence readout (FLRO) of 3 phases (1-120 s; 121-240 s; 241-360 s) was generated for 5 individual joints of the clinical predominant hand (carpal joint, metacarpophalangeal and proximal interphalangeal joints of digits II & III). To dissect the effect of the overall perfusion of the hand from the perfusion due to synovitis, a fluorescence ratio (FLRA) was additionally calculated, dividing each FLRO by the readout of the eponychium of digit II. The mean FLRO and FLRA were compared between joints with absent vs. present synovitis determined by clinical examination, grayscale, color Doppler ultrasonography, or magnetic resonance imaging (MRI).

Results

The analysis for 90 individual joints from 18 patients yielded FLRO ranging from 4.4 to 49.0 × 10³, and FLRAs ranging from 0.37 to 2.27. Overall, the analyses based on the FLRA revealed a higher discrimination than the analyses related to the FLRO, demonstrating most significant differences in phases 2 and 3. A sensitivity of 26/39 (67%) and a specificity of 31/40 (77%) were calculated for FLRA of phase 3 using a cut-off value of more than 1.2 to detect MRI-confirmed synovitis with FOI.

Conclusions

FOI has a potential for visualizing synovitis in subjects with RA. For adequate FOI interpretation, quantitative analysis should be based on the novel FLRA calculated for phases 2 and 3.     

Subclinical inflammation on MRI of hand and foot of anticitrullinated peptide antibody–negative arthralgia patients at risk for rheumatoid arthritis

Introduction

It is known that anticitrullinated peptide antibody (ACPA)–positive rheumatoid arthritis (RA) has a preclinical phase. Whether this phase is also present in ACPA-negative RA is unknown. To determine this, we studied ACPA-negative arthralgia patients who were considered prone to progress to RA for local subclinical inflammation observed on hand and foot magnetic resonance imaging (MRI) scans.

Methods

We studied a total of 64 ACPA-negative patients without clinically detectable arthritis and with arthralgia of the small joints within the previous 1 year. Because of the character of the patients’ symptoms, the rheumatologists considered these patients to be prone to progress to RA. For comparisons, we evaluated 19 healthy, symptom-free controls and 20 ACPA-negative RA patients, who were identified according to the 1987 American Rheumatism Association criteria. All participants underwent MRI of unilateral wrist, metacarpophalangeal and metatarsophalangeal joints. Synovitis and bone marrow oedema (BME) were scored according to the OMERACT rheumatoid arthritis magnetic resonance imaging scoring system, and the scores were summed to yield the ‘MRI inflammation score’. Scores were compared between groups. Among the ACPA-negative arthralgia patients, MRI inflammation scores were related to C-reactive protein (CRP) levels and the tenderness of scanned joints.

Results

MRI inflammation scores increased progressively among the groups of controls and ACPA-negative arthralgia and RA patients (median scores = 0, 1 and 10, respectively; P < 0.001). The MRI inflammation scores of ACPA-negative arthralgia patients were significantly higher than those of controls (P = 0.018). In particular, the synovitis scores were higher in ACPA-negative arthralgia patients (P = 0.046). Among the ACPA-negative arthralgia patients, inflammation was observed predominantly in the wrist (53%). The synovitis scores were associated with CRP levels (P = 0.007) and joint tenderness (P = 0.026). Despite the limited follow-up duration, five patients developed clinically detectable arthritis. These five patients had higher scores for MRI inflammation (P = 0.001), synovitis (P = 0.002) and BME (P = 0.003) compared to the other patients.

Conclusion

Subclinical synovitis was observed in the small joints of ACPA-negative arthralgia patients, and especially in patients whose conditions progressed to clinically detectable arthritis. This finding suggests the presence of a preclinical phase in ACPA-negative RA. Further longitudinal studies of these lesions and patients are required to confirm this hypothesis.     

Cholesterol accumulation caused by low density lipoprotein receptor deficiency or a cholesterol-rich diet results in ectopic bone formation during experimental osteoarthritis

Introduction

Osteoarthritis (OA) is associated with the metabolic syndrome, however the underlying mechanisms remain unclear. We investigated whether low density lipoprotein (LDL) accumulation leads to increased LDL uptake by synovial macrophages and affects synovial activation, cartilage destruction and enthesophyte/osteophyte formation during experimental OA in mice.

Methods

LDL receptor deficient (LDLr^−/−) mice and wild type (WT) controls received a cholesterol-rich or control diet for 120 days. Experimental OA was induced by intra-articular injection of collagenase twelve weeks after start of the diet. OA knee joints and synovial wash-outs were analyzed for OA-related changes. Murine bone marrow derived macrophages were stimulated with oxidized LDL (oxLDL), whereupon growth factor presence and gene expression were analyzed.

Results

A cholesterol-rich diet increased apolipoprotein B (ApoB) accumulation in synovial macrophages. Although increased LDL levels did not enhance thickening of the synovial lining, S100A8 expression within macrophages was increased in WT mice after receiving a cholesterol-rich diet, reflecting an elevated activation status. Both a cholesterol-rich diet and LDLr deficiency had no effect on cartilage damage; in contrast, ectopic bone formation was increased within joint ligaments (fold increase 6.7 and 6.1, respectively). Moreover, increased osteophyte size was found at the margins of the tibial plateau (4.4 fold increase after a cholesterol-rich diet and 5.3 fold increase in LDLr^−/− mice). Synovial wash-outs of LDLr^−/− mice and supernatants of macrophages stimulated with oxLDL led to increased transforming growth factor-beta (TGF-β) signaling compared to controls.

Conclusions

LDL accumulation within synovial lining cells leads to increased activation of synovium and osteophyte formation in experimental OA. OxLDL uptake by macrophages activates growth factors of the TGF-superfamily.     

Interleukin-10 regulates the inflammasome-driven augmentation of inflammatory arthritis and joint destruction

Introduction

Activation of the inflammasome has been implicated in the pathology of various autoinflammatory and autoimmune diseases. While the NLRP3 inflammasome has been linked to arthritis progression, little is known about its synovial regulation or contribution to joint histopathology. Regulators of inflammation activation, such as interleukin (IL)-10, may have the potential to limit the inflammasome-driven arthritic disease course and associated structural damage. Hence, we used IL-10-deficient (IL-10KO) mice to assess NLRP3 inflammasome-driven arthritic pathology.

Methods

Antigen-induced arthritis (AIA) was established in IL-10KO mice and wild-type controls. Using histological and radiographic approaches together with quantitative real-time PCR of synovial mRNA studies, we explored the regulation of inflammasome components. These were combined with selective blocking agents and ex vivo investigative studies in osteoclast differentiation assays.

Results

In AIA, IL-10KO mice display severe disease with increased histological and radiographic joint scores. Here, focal bone erosions were associated with increased tartrate-resistant acid phosphatase (TRAP)-positive cells and a localized expression of IL-1β. When compared to controls, IL-10KO synovium showed increased expression of Il1b, Il33 and NLRP3 inflammasome components. Synovial Nlrp3 and Casp1 expression further correlated with Acp5 (encoding TRAP), while neutralization of IL-10 receptor signaling in control mice caused increased expression of Nlrp3 and Casp1. In ex vivo osteoclast differentiation assays, addition of exogenous IL-10 or selective blockade of the NLRP3 inflammasome inhibited osteoclastogenesis.

Conclusions

These data provide a link between IL-10, synovial regulation of the NLRP3 inflammasome and the degree of bone erosions observed in inflammatory arthritis.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0419-y) contains supplementary material, which is available to authorized users.     

MicroPure Imaging for the Evaluation of Microcalcifications in Gouty Arthritis Involving the First Metatarsophalangeal Joint: A Preliminary Study

Objective

To assess the value of MicroPure, a new ultrasound image processing technique, in identifying microcalcifications (formed by monosodium urate crystals) in the first metatarsophalangeal joints attacked by gout compared to gray-scale ultrasound images.

Materials and Methods

Thirty-six patients who fulfilled the study inclusion criteria underwent gray-scale ultrasound and MicroPure examinations of the first metatarsophalangeal joints attacked by gout. Static images of the target areas were acquired using gray-scale ultrasound and MicroPure. Two independent and blinded investigators analyzed the images to determine the number of microcalcifications and to score for image quality and artifacts.

Results

The two investigators observed significantly more microcalcifications with MicroPure compared to gray-scale ultrasound (ρ<0.001). The level of agreement between the investigators consistently increased from gray-scale ultrasound to MicroPure imaging (gray-scale interclass correlation coefficient of 0.69 vs. MicroPure interclass correlation coefficient of 0.81). One investigator preferred the MicroPure image quality over gray-scale ultrasound (ρ<0.001), but the other investigator disagreed (ρ<0.001). Both investigators observed fewer artifacts with MicroPure than with gray-scale ultrasound (ρ<0.009).

Conclusion

MicroPure imaging identified significantly more microcalcifications than gray-scale ultrasound.     

Acute-Phase Serum Amyloid A in Osteoarthritis: Regulatory Mechanism and Proinflammatory Properties

Objective

To determine if serum amyloid A (A-SAA) could be detected in human osteoarthritic (OA) joints and further clarify if high A-SAA level in joints result from a local production or from a diffusion process from abnormally elevated plasma concentration. Regulatory mechanism of A-SAA expression and its pro-inflammatory properties were also investigated.

Methods

A-SAA levels in serum and synovial fluid of OA (n = 29) and rheumatoid arthritis (RA) (n = 27) patients were measured and compared to matched-healthy volunteers (HV) (n = 35). In vitro cell cultures were performed on primary joint cells provided from osteoarthritis patients. Regulatory mechanisms were studied using Western-blotting, ELISA and lentiviral transfections.

Results

A-SAA was statistically increased in OA plasma patients compared to HV. Moreover, A-SAA level in OA plasma and synovial fluid increased with the Kellgren & Lauwrence grade. For all OA and RA patients, A-SAA plasma level was higher and highly correlated with its corresponding level in the synovial fluid, therefore supporting that A-SAA was mainly due to the passive diffusion process from blood into the joint cavity. However, A-SAA expression was also observed in vitro under corticosteroid treatment and/or under IL-1beta stimuli. A-SAA expression was down-regulated by PPAR-γ agonists (genistein and rosiglitazone) and up-regulated by TGF-β1 through Alk1 (Smad1/5) pathway. RhSAA induced proinflammatory cytokines (IL-6, IL-8, GRO-α and MCP-1) and metalloproteinases (MMP-1, MMP-3 and MMP-13) expression in FLS and chondrocytes, which expression was downregulated by TAK242, a specific TLR4 inhibitor.

Conclusion

Systemic or local A-SAA expression inside OA joint cavity may play a key role in inflammatory process seen in osteoarthritis, which could be counteracted by TLR4 inhibition.     

Diagnostic utility of musculoskeletal ultrasound in patients with suspected arthritis – a probabilistic approach

Introduction

This study aimed to assess the utility of musculoskeletal ultrasound (MSUS) in patients with joint symptoms using a probabilistic approach.

Methods

One hundred and three patients without prior rheumatologic diagnosis and referred to our clinic for evaluation of inflammatory arthritis were included. Patients were assessed clinically including joint examination, laboratory testing including acute-phase reactants, rheumatoid factor (RF) and anti citrulinated protein antibody (ACPA), and radiographs of hands and feet if clinically indicated. A diagnostic assessment was then performed by the responsible rheumatologist where the probability of a) any inflammatory arthritis and b) rheumatoid arthritis was given on a 5-point scale ranging from 0 to 20% up to 80 to 100% probability. Subsequently, an ultrasound examination of the wrist, metacarpophalangeal (MCP), proximal interphalangeal (PIP) joints 2 to 5 in both hands, metatarsophalangeal (MTP) joints 2 to 5 in both feet and any symptomatic joints was performed and the results presented to the same rheumatologist. The latter then assessed the diagnostic probabilities again, using the same scale.

Results

The rheumatologists’ certainty for presence/absence of inflammatory arthritis and rheumatoid arthritis was increased significantly following ultrasound performance. The proportion of patient for whom diagnostic certainty for inflammatory arthritis was maximal was 33.0% before and 71.8% after musculoskeletal ultrasound (P <0.001). With regard to a diagnosis of RA, the proportions were 31.1% pre-test and 61.2% post-test (P <0.001). MSUS findings agreed with the final diagnosis in 95% of patients.

Conclusion

Musculoskeletal ultrasound, when added to routine rheumatologic investigation, greatly increases the diagnostic certainty in patients referred for the evaluation of inflammatory arthritis. The changes from pre-test to post-test probability quantify the diagnostic utility of musculoskeletal ultrasound in probabilistic terms.     

Blockade of IL-36 Receptor Signaling Does Not Prevent from TNF-Induced Arthritis

Introduction

Interleukin (IL)-36α is a newly described member of the IL-1 cytokine family with a known inflammatory and pathogenic function in psoriasis. Recently, we could demonstrate that the receptor (IL-36R), its ligand IL-36α and its antagonist IL-36Ra are expressed in synovial tissue of arthritis patients. Furthermore, IL-36α induces MAP-kinase and NFκB signaling in human synovial fibroblasts with subsequent expression and secretion of pro-inflammatory cytokines.

Methods

To understand the pathomechanism of IL-36 dependent inflammation, we investigated the biological impact of IL-36α signaling in the hTNFtg mouse. Also the impact on osteoclastogenesis by IL-36α was tested in murine and human osteoclast assays.

Results

Diseased mice showed an increased expression of IL-36R and IL-36α in inflamed knee joints compared to wildtype controls. However, preventively treating mice with an IL-36R blocking antibody led to no changes in clinical onset and pattern of disease. Furthermore, blockade of IL-36 signaling did not change histological signs of TNF-induced arthritis. Additionally, no alteration on bone homeostasis was observed in ex vivo murine and human osteoclast differentiation assays.

Conclusion

Thus we conclude that IL-36α does not affect the development of inflammatory arthritis.     

Demonstration of extracellular peptidylarginine deiminase (PAD) activity in synovial fluid of patients with rheumatoid arthritis using a novel assay for citrullination of fibrinogen

Introduction

Members of the peptidylarginine deiminase (PAD) family catalyse the posttranslational conversion of peptidylarginine to peptidylcitrulline. Citrullination of proteins is well described in rheumatoid arthritis (RA), and hypercitrullination of proteins may be related to inflammation in general. PAD activity has been demonstrated in various cell lysates, but so far not in synovial fluid. We aimed to develop an assay for detection of PAD activity, if any, in synovial fluid from RA patients.

Methods

An enzyme-linked immunosorbent assay using human fibrinogen as the immobilized substrate for citrullination and anti-citrullinated fibrinogen antibody as the detecting agent were used for measurement of PAD activity in synovial fluid samples from five RA patients. The concentrations of PAD2 and calcium were also determined.

Results

Approximately 150 times lower levels of recombinant human PAD2 (rhPAD2) than of rhPAD4 were required for citrullination of fibrinogen. PAD activity was detected in four of five synovial fluid samples from RA patients and correlated with PAD2 concentrations in the samples (r = 0.98, P = 0.003). The calcium requirement for half-maximal activities of PAD2 and PAD4 were found in a range from 0.35 to 1.85 mM, and synovial fluid was found to contain sufficient calcium levels for the citrullination process to occur.

Conclusions

We present an assay with high specificity for PAD2 activity and show that citrullination of fibrinogen can occur in cell-free synovial fluid from RA patients.     

Clinical characteristics in subjects with NLRP3 V198M diagnosed at a single UK center and a review of the literature

Introduction

Mutations in the NLRP3 gene are associated with the dominantly inherited cryopyrin-associated periodic syndrome (CAPS). The significance of the V198M variant is unclear; it has been reported in association with various CAPS phenotypes and as a variant of uncertain consequence. The aim of this study was to characterize the clinical phenotypes and treatments in individuals with V198M assessed in a single UK center.

Methods

DNA samples from 830 subjects with fever syndromes or a family history of CAPS were screened for mutations in the NLRP3 gene with polymerase chain reaction (PCR) and sequencing. A detailed medical history was available in all cases. Inflammatory disease activity was monitored monthly with measurements of serum amyloid A protein (SAA) and C-reactive protein (CRP) in symptomatic individuals.

Results

NLRP3 V198M was identified in 19 subjects. It was found in association with CAPS in five cases, in one patient with Schnitzler syndrome, in three patients who also had a nucleotide alteration in another fever gene, and in three other patients with evidence of an autoinflammatory phenotype. Seven asymptomatic individuals were detected during screening of family members.

Conclusions

The NLRP3 V198M variant shows variable expressivity and reduced penetrance. It may be associated with classical inherited or apparently sporadic CAPS and with atypical autoinflammatory disease of varying severity, intriguingly including Schnitzler syndrome. The factors that influence the pathogenic consequences of this variant remain unknown. However, the remarkable response to interleukin 1 (IL-1) blockade in all but one individual in our series confirms that their clinical features are indeed mediated by IL-1.     

Gene expression and activity of cartilage degrading glycosidases in human rheumatoid arthritis and osteoarthritis synovial fibroblasts

Introduction

Similar to matrix metalloproteinases, glycosidases also play a major role in cartilage degradation. Carbohydrate cleavage products, generated by these latter enzymes, are released from degrading cartilage during arthritis. Some of the cleavage products (such as hyaluronate oligosaccharides) have been shown to bind to Toll-like receptors and provide endogenous danger signals, while others (like N-acetyl glucosamine) are reported to have chondroprotective functions. In the current study for the first time we systematically investigated the expression of glycosidases within the joints.

Methods

Expressions of β-D-hexosaminidase, β-D-glucuronidase, hyaluronidase, sperm adhesion molecule 1 and klotho genes were measured in synovial fibroblasts and synovial membrane samples of patients with rheumatoid arthritis and osteoarthritis by real-time PCR. β-D-Glucuronidase, β-D-glucosaminidase and β-D-galactosaminidase activities were characterized using chromogenic or fluorogenic substrates. Synovial fibroblast-derived microvesicles were also tested for glycosidase activity.

Results

According to our data, β-D-hexosaminidase, β-D-glucuronidase, hyaluronidase, and klotho are expressed in the synovial membrane. Hexosaminidase is the major glycosidase expressed within the joints, and it is primarily produced by synovial fibroblasts. HexA subunit gene, one of the two genes encoding for the alpha or the beta chains of hexosaminidase, was characterized by the strongest gene expression. It was followed by the expression of HexB subunit gene and the β-D-glucuronidase gene, while the expression of hyaluronidase-1 gene and the klotho gene was rather low in both synovial fibroblasts and synovial membrane samples. Tumor growth factor-β1 profoundly downregulated glycosidase expression in both rheumatoid arthritis and osteoarthritis derived synovial fibroblasts. In addition, expression of cartilage-degrading glycosidases was moderately downregulated by proinflammatory cytokines including TNFα, IL-1β and IL-17.

Conclusions

According to our present data, glycosidases expressed by synovial membranes and synovial fibroblasts are under negative regulation by some locally expressed cytokines both in rheumatoid arthritis and osteoarthritis. This does not exclude the possibility that these enzymes may contribute significantly to cartilage degradation in both joint diseases if acting in collaboration with the differentially upregulated proteases to deplete cartilage in glycosaminoglycans.