Marleen G. H. van de Sande

Interleukin-17–positive mast cells contribute to synovial inflammation in spondylarthritis

OBJECTIVE Studies comparing spondylarthritis (SpA) to rheumatoid arthritis (RA) synovitis suggest that innate immune cells may play a predominant role in the pathogenesis of SpA. Recent observations have indicated a marked synovial mast cell infiltration in psoriatic SpA. We therefore undertook the present study to investigate the potential contribution of mast cells to synovial inflammation in SpA. METHODS Synovial tissue and fluid were obtained from patients with either nonpsoriatic or psoriatic SpA (n=82) and patients with RA (n=50). Synovial biopsy tissue was analyzed by immunostaining and used in ex vivo cultures. Synovial fluid was analyzed by enzyme-linked immunosorbent assay. RESULTS We observed a strong and specific increase of c-Kit-positive mast cells in the synovium from patients with SpA compared to the synovium from patients with RA synovitis, which was independent of disease subtype (nonpsoriatic versus psoriatic), disease duration, and treatment. Staining of mast cell granules, analysis of synovial fluid, and results in ex vivo tissue culture did not indicate increased degranulation in SpA synovitis. However, mast cells expressed significantly more interleukin-17 (IL-17) in SpA than in RA synovitis, and mast cells constituted the major IL-17-expressing cell population in the SpA synovium. Ex vivo targeting of synovial mast cells with the c-Kit inhibitor imatinib mesylate significantly decreased the production of IL-17 as well as other proinflammatory cytokines in synovial tissue cultures. Analysis of paired pre- and posttreatment synovial tissue samples indicated that the mast cell/IL-17 axis in SpA was not modulated by effective tumor necrosis factor (TNF) blockade. CONCLUSION The specific and TNF-independent increase in IL-17-expressing mast cells may contribute to the progression of synovial inflammation in peripheral SpA.

Smoking and overweight determine the likelihood of developing rheumatoid arthritis

Objectives Rheumatoid arthritis (RA) is a prototypic chronic inflammatory disease with a debilitating course if untreated. A genetic predisposition for RA is known, and its occurrence is associated with the presence of autoantibodies in the serum and with environmental factors. It is unknown if smoking and overweight are contributory factors for developing RA in individuals with RA-specific autoantibodies in the serum. Methods Fifty-five individuals at risk for developing RA, based on the presence of RA-specific autoantibodies in the serum, who never had any evidence of arthritis upon physical examination, were followed over time. Smoking was assessed as being never or ever smoker and body mass index as <25 (normal) or ≥25 kg/m2 (overweight). Clinical endpoint was the occurrence of arthritis. Proportional hazard regression analysis was performed to investigate the potential of (combinations of) variables in predicting the onset of arthritis over time. Results After a median follow up time of 13 (IQR 6–27) months, 15 individuals (27%) developed arthritis. Smoking was associated with the development of arthritis (HR (95% CI): 9.6 (1.3 to 73.0); p=0.029). Overweight was, independently of smoking, associated with arthritis (HR (95% CI): 5.6 (1.3 to 25.0); p=0.023). The overall arthritis risk of 28% after a median of 27 months follow up increased to 60% in individuals with a smoking history combined with overweight. Conclusions This is the first prospective study showing that smoking and overweight increase the risk of development of arthritis in a cohort of autoantibody-positive individuals at risk for developing RA. These results show the importance of life style factors in development of RA and should be critically evaluated in future clinical research aimed at disease prevention.

Evaluating antirheumatic treatments using synovial biopsy: a recommendation for standardisation to be used in clinical trials

Inflammation of synovium is one of the hallmarks of rheumatoid arthritis (RA). Analysis of synovial tissue has increased our understanding of RA pathogenesis, aided in identifying potential therapeutic targets and has been used in the response and mechanistic evaluation of antirheumatic treatments. In addition, studies are ongoing, aimed at the identification of diagnostic and prognostic biomarkers in the synovium. This paper outlines the currently used procedures for sampling and processing of synovial tissue, and presents a standardised recommendation to support multicentre translational research.

Synovial tissue sublining CD68 expression is a biomarker of therapeutic response in rheumatoid arthritis clinical trials: Consistency across centers

Objective. To determine whether the correlation between the mean change in disease activity and the mean change in synovial sublining (sl) CD68 expression could be demonstrated across different academic centers. Methods. Synovial biopsies obtained at arthroscopy from patients with rheumatoid arthritis before and 160 days after rituximab therapy were selected and coded. Paired sections were processed independently at Amsterdam Medical Center (AMC) and at St. Vincent’s University Hospital (SVUH), Dublin. Digital image analysis (DIA) was employed at both centers to quantify sublining CD68 expression. Results. After analysis of CD68sl expression at centers in 2 different countries, high levels of intracenter and intercenter agreement were observed. For the pooled sections stained at AMC, the correlation between 2 investigators was R = 0.942, p = 0.000, and for sections stained at SVUH, R = 0.899, p = 0.001. Similarly, the intracenter correlations for ΔCD68sl expression after treatment were R = 0.998, p = 0.000, for sections stained at AMC and R = 0.880, p = 0.000, for sections stained at SVUH. The intercenter correlation for the pooled scores of sections stained at AMC was R = 0.85, p = 0.000, and for the sections stained at SVUH, R = 0.62, p = 0.001. The consistent correlation between ΔDAS (Disease Activity Score) and ΔCD68sl expression across different studies (Pearson correlation = 0.895, p < 0.001) was confirmed. The standardized response mean values for ΔCD68sl, calculated from analyses at both AMC and SVUH, were consistently 0.5 or greater, indicating a moderate to high potential to detect change. Conclusion. The correlation between mean ΔDAS and mean ΔCD68sl expression was confirmed across 2 centers. Examination of serial biopsy samples can be used reliably to screen for interesting biological effects at the site of inflammation at an early stage of drug development.

Rheumatoid Synovial Inflammation: Pixel-by-Pixel Dynamic Contrast-enhanced MR Imaging Time-Intensity Curve Shape Analysis—A Feasibility Study

PURPOSE To analyze the distribution of different shapes of time-intensity curves (TICs) in synovial tissue of patients with rheumatoid arthritis (RA) and to compare relative numbers of TIC shapes between patients with RA and healthy control subjects. MATERIALS AND METHODS This prospective study was approved by the institutional review board; patients and control subjects gave written informed consent. Dynamic contrast material-enhanced magnetic resonance (MR) imaging of the knee joint in five patients with early RA and in five control subjects was performed. Parametric maps showing seven TIC shape types were created. Spatial information of the synovial TIC shape distribution pattern and relative number of TIC shapes were calculated on a three-dimensional region of interest. Relative TIC shape numbers were compared by using a nonparametric Mann-Whitney U test. RESULTS Synovial enhancement in patients with RA consisted of type 2 TIC shapes (slow enhancement) with heterogeneous zones of types 3 (fast enhancement followed by plateau phase), 4 (fast enhancement followed by early washout phase), and 5 (fast enhancement followed by slow enhancement increase) TIC shapes, compared with almost only type 2 TIC shapes in control subjects. The heterogeneous zones were seen in the lateral and medial knee compartments and around the cruciate ligaments. A significantly higher relative number of type 4 TIC shapes was observed in the patient group compared with the control group (16.5% vs 6.9%, P = .008). CONCLUSION The pixel-by-pixel dynamic contrast-enhanced MR imaging TIC shape analysis may help distinguish patients with RA from control subjects on the basis of the relative number of type 4 TIC shapes. This study provides the rationale for future research to evaluate the utility of this approach in clinical practice.

Selective involvement of ERK and JNK mitogen-activated protein kinases in early rheumatoid arthritis (1987 ACR criteria compared to 2010 ACR/EULAR criteria): a prospective study aimed at identification of diagnostic and prognostic biomarkers as well as therapeutic targets

Objectives To investigate the expression and activation of mitogen-activated protein kinases in patients with early arthritis who are disease-modifying antirheumatic drug (DMARD) naïve. Methods A total of 50 patients with early arthritis who were DMARD naïve (disease duration <1 year) were prospectively followed and diagnosed at baseline and after 2 years for undifferentiated arthritis (UA), rheumatoid arthritis (RA) (1987 American College of Rheumatology (ACR) and 2010 ACR/European League Against Rheumatism (EULAR) criteria), or spondyloarthritis (SpA). Synovial biopsies obtained at baseline were examined for expression and phosphorylation of p38, extracellular signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) by immunohistochemistry and digital analysis. Synovial tissue mRNA expression was measured by quantitative PCR (qPCR). Results ERK and JNK activation was enhanced at inclusion in patients meeting RA criteria compared to other diagnoses. JNK activation was enhanced in patients diagnosed as having UA at baseline who eventually fulfilled 1987 ACR RA criteria compared to those who remained UA, and in patients with RA fulfilling 2010 ACR/EULAR criteria at baseline. ERK and JNK activation was enhanced in patients with RA developing progressive joint destruction. JNK activation in UA predicted 1987 ACR RA classification criteria fulfilment (R2=0.59, p=0.02) after follow-up, and disease progression in early arthritis (R2=0.16, p<0.05). Enhanced JNK activation in patients with persistent disease was associated with altered synovial expression of extracellular matrix components and CD44. Conclusions JNK activation is elevated in RA before 1987 ACR RA classification criteria are met and predicts development of erosive disease in early arthritis, suggesting JNK may represent an attractive target in treating RA early in the disease process.

Disease-specific and inflammation-independent stromal alterations in spondylarthritis synovitis

OBJECTIVE The molecular processes driving the distinct patterns of synovial inflammation and tissue remodeling in spondylarthritis (SpA) as compared to rheumatoid arthritis (RA) remain largely unknown. Therefore, we aimed to identify novel and unsuspected disease-specific pathways in SpA by a systematic and unbiased synovial gene expression analysis. METHODS Differentially expressed genes were identified by pan-genomic microarray and confirmed by quantitative polymerase chain reaction and immunohistochemical analyses of synovial tissue biopsy samples from patients with SpA (n=63), RA (n=28), and gout (n=9). The effect of inflammation on gene expression was assessed by stimulating fibroblast-like synoviocytes (FLS) with synovial fluid and by analysis of synovial tissue samples at weeks 0 and 12 of etanercept treatment. RESULTS Using very stringent statistical thresholds, microarray analysis identified 64 up-regulated transcripts in patients with SpA synovitis as compared to those with RA synovitis. Pathway analysis revealed a robust myogene signature in this gene set. The myogene signature was technically and biologically reproducible, was specific for SpA, and was independent of disease duration, treatment, and SpA subtype (nonpsoriatic versus psoriatic). Synovial tissue staining identified the myogene expressing cells as vimentin-positive, prolyl 4-hydroxylase β-positive, CD90+, and CD146+ mesenchymal cells that were significantly overrepresented in the intimal lining layer and synovial sublining of inflamed SpA synovium. Neither in vitro exposure to synovial fluid from inflamed SpA joints nor in vivo blockade of tumor necrosis factor modulated the SpA-specific myogene signature. CONCLUSION These data identify a novel and disease-specific myogene signature in SpA synovitis. The fact that this stromal alteration appeared not to be downstream of local inflammation warrants further analysis of its functional role in the pathogenesis of the disease.

Presence of lymphocyte aggregates in the synovium of patients with early arthritis in relationship to diagnosis and outcome: is it a constant feature over time?

Objectives To evaluate the presence of lymphocyte aggregates in synovial tissue of patients with early arthritis in relationship to clinical outcome and to determine whether this is a stable feature over time. Methods Arthroscopic synovial biopsy samples were collected in a prospective cohort of disease-modifying antirheumatic drug-naïve patients with early arthritis (<1 years disease duration) at baseline (n=93) and, if rheumatoid arthritis was suspected, after 6 months of follow-up (n=17). After 2 years of follow-up, definitive diagnosis and clinical outcome were assessed. Size of synovial lymphocyte aggregates was graded (score 1–3). Lymphoid neogenesis (LN) was defined by the presence of grade ≥2 aggregates and subclassified based on the presence of follicular dendritic cells (FDCs). Results LN was present in 36% of all patients and FDCs in 15% of patients with LN. Presence of lymphocyte aggregates differed over time. LN was associated with the degree of synovial inflammation. There was no relationship between the presence of lymphocyte aggregates at baseline and definitive diagnosis or clinical outcome after follow-up. Conclusions Presence of lymphocyte aggregates is a dynamic phenomenon related to the degree of synovitis and can be detected in different forms of early arthritis. This feature does not appear to be related to clinical outcome.

Characteristics of synovial inflammation in early arthritis analysed by pixel-by-pixel time-intensity curve shape analysis

OBJECTIVE Dynamic contrast-enhanced (DCE-MRI) time-intensity curve (TIC) shape analysis has previously been used in oncology, where fast initial enhancement and early washout are associated with malignancy. As RA synovium has some tumour-like features, we investigated DCE-MRI TIC shape expression in early arthritis in relationship to diagnosis. METHODS Twenty-eight DMARD-naïve, early arthritis patients (<1 year) with inflammation of at least one knee joint were included. At baseline DCE-MRI of the inflamed knee joint was performed, and the TIC shape type expression, maximal enhancement, maximum slope of increase and total volume of enhancing pixels were calculated. In addition, disease activity parameters were determined. At 2 years of follow-up, patients were classified as RA or non-RA according to established classification criteria. RESULTS Type 4 TIC shape, characterized by fast initial enhancement followed by a quick washout phase, was significantly higher in patients fulfilling classification criteria for RA after 2 years of follow-up compared with non-RA patients (15.6 and 7.9%, respectively, P = 0.02). All other DCE-MRI parameters showed no differences between the groups, highlighting the specificity of this observation. CONCLUSION A high expression of aggressive DCE-MRI TIC shape Type 4 is associated with RA. Our results are consistent with the view that increased vascularity plays a key role in the pathogenesis of RA. This study underlines the rationale for further studies investigating the prospect of DCE-MRI TIC shape analysis as a diagnostic tool in early arthritis and the relationship with development of destructive disease.

Hunting for the pathogenesis of rheumatoid arthritis: core-needle biopsy of inguinal lymph nodes as a new research tool

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease affecting mainly the synovial joints. Processes involved in the initiation of inflammation in RA are largely unknown. Recent work has shown that systemic autoimmunity precedes synovial inflammation in autoantibody positive individuals developing RA1 and animal models have suggested that lymph node changes may precede inflammation in the synovial tissue.2 Ultrasound guided lymph node biopsy under local anaesthesia has previously been used as a diagnostic tool in haematology and oncology.3 Therefore, we started to use this technique to provide insight into the pathogenic processes involved in the earliest phases of RA, by analysing lymph node tissue obtained during different phases of the disease. Our experience includes use of this technique in autoantibody positive individuals without arthritis who are at risk of developing RA,4 early arthritis patients and established RA …