Expression of 5-lipoxygenase and 15-lipoxygenase in rheumatoid arthritis synovium and effects of intraarticular glucocorticoids

The past years have witnessed tremendous progress in the treatment of rheumatoid arthritis, a chronic debilitating autoimmune disease mainly characterized by joint inflammation with progressive tissue destruction and loss of function. This condition affects 0.5-1% of the population, is associated with important co-morbidities and represents a heavy economical burden. New strategies, employing early and aggressive therapies with classical drugs or new agents, have resulted in impressive improvements in controlling disease activity. In some cases they even lead to clinical remission. Despite potent and efficient biological agents that specifically modulate distinct pathological pathways a large proportion of patients remain unresponsive to these therapies; drug-free remission is also difficult to achieve since attempting discontinuation of treatment usually results in disease flare. In rheumatoid arthritis joints there is a constant activation of complex networks of cytokines and factors mediating immune interactions and inflammation, in which prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) are important players and contributors to pathogenesis. Our research aimed to investigate the synovial expression of enzymes controlling prostaglandin E2 synthesis and degradation – cyclooxygenase (COX) 1 and 2, microsomal prostaglandin E2 synthase 1 (MPGES1) and 15-prostaglandin dihydrogenase (15-PGDH) as well as enzymes involved in leukotriene synthesis, such as 5-lipoxygenase (LO) and 15-LO. In addition, we evaluated how traditional and new therapies influence these pathways, by analyzing enzyme expression before and after systemic treatment with tumor necrosis factor (TNF) antagonists, rituximab or methotrexate, as well as before and after intra-articular treatment with glucocorticoids. We also evaluated the in vitro effects of TNF antagonists and glucocorticoids on synovial fluid cells and that of methotrexate on synovial fibroblasts. We demonstrated that synovial tissue from RA patients displayed an important expression of enzymes involved in the metabolism of PGE2, as well as 5-LO and 15-LO. MPGES1 and COX-2, the inflammation-inducible enzymes co-localized mainly in fibroblasts and macrophage-like cells and accounted for the local PGE2 production. Intra-articular glucocorticoids significantly reduced all enzymes involved in the PGE2 cascade – COX-1 and COX-2, MPGES1 and 15-PGDH, but also 5-LO, responsible for leukotriene formation. However, they did not influence the expression of 15-LO, an enzyme involved in the formation of both pro-and anti-inflammatory lipid mediators. Regarding the effects of TNF blockers, rituximab or methotrexate, they did not alter the expression profile of enzymes involved in PGE2 metabolism despite showing clinical efficiency in improving disease activity. Although anti-TNF agents reduced the in vitro expression of MPGES1 and COX-2 in synovial fluid cells, the lack of effect ex vivo in biopsies emphasized once again the differences between synovial compartments and possibly the difficulty in mimicking the micro-environment at the site of inflammation in vitro. In conclusion, this thesis demonstrates that potent anti-rheumatic drugs currently used in the clinic with good efficiency also leave inflammatory pathways un-affected, which may account for subclinical ongoing disease activity. Blocking the PGE2 pathway by using MPGES1 inhibitors as combination therapy may show benefit in dampening ongoing local inflammation

Variability in synovial inflammation in rheumatoid arthritis investigated by microarray technology

In recent years microarray technology has been used increasingly to acquire knowledge about the pathogenic processes involved in rheumatoid arthritis. The present study investigated variations in gene expression in synovial tissues within and between patients with rheumatoid arthritis. This was done by applying microarray technology on multiple synovial biopsies obtained from the same knee joints. In this way the relative levels of intra-patient and inter-patient variation could be assessed. The biopsies were obtained from 13 different patients: 7 by orthopedic surgery and 6 by rheumatic arthroscopy. The data show that levels of heterogeneity varied substantially between the biopsies, because the number of genes found to be differentially expressed between pairs of biopsies from the same knee ranged from 6 to 2,133. Both arthroscopic and orthopedic biopsies were examined, allowing us to compare the two sampling methods. We found that the average number of differentially expressed genes between biopsies from the same patient was about three times larger in orthopedic than in arthroscopic biopsies. Using a parallel analysis of the tissues by immunohistochemistry, we also identified orthopedic biopsies that were unsuitable for gene expression analysis of synovial inflammation due to sampling of non-inflamed parts of the tissue. Removing these biopsies reduced the average number of differentially expressed genes between the orthopedic biopsies from 455 to 171, in comparison with 143 for the arthroscopic biopsies. Hierarchical clustering analysis showed that the remaining orthopedic and arthroscopic biopsies had gene expression signatures that were unique for each patient, apparently reflecting patient variation rather than tissue heterogeneity. Subsets of genes found to vary between biopsies were investigated for overrepresentation of biological processes by using gene ontology. This revealed representative 'themes' likely to vary between synovial biopsies affected by inflammatory disease

Evaluation of arthroscopy and macroscopic scoring

INTRODUCTION: Arthroscopy is a minimally invasive technique for retrieving synovial biopsies in rheumatology during the past 20 years. Vital for its use is continual evaluation of its safety and efficacy. Important for sampling is the fact of intraarticular variation for synovial markers. For microscopic measurements scoring systems have been developed and validated, but for macroscopic evaluations there is a need for further comprehensive description and validation of equivalent scoring systems. METHODS: We studied the complication rate and yield of arthroscopies performed at our clinic between 1998 and 2005. We also created and evaluated a macroscopic score set of instructions for synovitis. RESULTS: Of 408 procedures, we had two major and one minor complication; two haemarthrosis and one wound infection, respectively. Pain was most often not a problem, but 12 procedures had to be prematurely ended due to pain. Yield of biopsies adequate for histology were 83% over all, 94% for knee joints and 34% for smaller joints. Video printer photographs of synovium taken during arthroscopy were jointly and individually reviewed by seven raters in several settings, and intra and inter rater variation was calculated. A macroscopic synovial scoring system for arthroscopy was created (Macro-score), based upon hypertrophy, vascularity and global synovitis. These written instructions were evaluated by five control-raters, and when evaluated individual parameters were without greater intra or inter rater variability, indicating that the score is reliable and easy to use. CONCLUSIONS: In our hands rheumatologic arthroscopy is a safe method with very few complications. For knee joints it is a reliable method to retrieve representative tissue in clinical longitudinal studies. We also created an easy to use macroscopic score, that needs to be validated against other methodologies. We hope it will be of value in further developing international standards in this area

Skewed distribution of proinflammatory CD4+CD28null T cells in rheumatoid arthritis

Expanded populations of CD4+ T cells lacking the co-stimulatory molecule CD28 (CD4+CD28null T cells) have been reported in several inflammatory disorders. In rheumatoid arthritis, increased frequencies of CD4+CD28null T cells in peripheral blood have previously been associated with extra-articular manifestations and human cytomegalovirus (HCMV) infection, but their presence in and contribution to joint manifestations is not clear. In the present article we investigated the distribution of CD4+CD28null T cells in the synovial membrane, synovial fluid and peripheral blood of RA patients, and analysed the association with erosive disease and anti-citrullinated protein antibodies. CD4+CD28null T cells were infrequent in the synovial membrane and synovial fluid, despite significant frequencies in the circulation. Strikingly, the dominant TCR-Vβ subsets of CD4+CD28null T cells in peripheral blood were often absent in synovial fluid. CD4+CD28null T cells in blood and synovial fluid showed specificity for HCMV antigens, and their presence was clearly associated with HCMV seropositivity but not with anti-citrullinated protein antibodies in the serum or synovial fluid, nor with erosive disease. Together these data imply a primary role for CD4+CD28null T cells in manifestations elsewhere than in the joints of patients with HCMV-seropositive rheumatoid arthritis

Tocilizumab decreases T cells but not macrophages in the synovium of patients with rheumatoid arthritis while it increases the levels of serum interleukin-6 and RANKL

Objectives Our knowledge about the effect of tocilizumab (TCZ) on the synovium in rheumatoid arthritis (RA) is limited. The aim of this study was to investigate the effect of TCZ on citrullination and on inflammation in the synovial tissue and in the peripheral blood.Methods 15 patients with RA underwent synovial biopsy before and 8 weeks after TCZ initiation. Clinical evaluation was performed at baseline and at 8 weeks. Using immunohistochemistry, we evaluated the expression of CD68, CD3, CD20, osteoprotegerin (OPG) and receptor activator for nuclear factor-κB ligand (RANKL) before and after treatment with TCZ. We also analysed the expression of protein arginine deiminase (PAD)-2 and PAD-4 enzymes in the synovial tissue and protein citrullination patterns with the help of anticitrullinated protein antibody (ACPA) clones 1325:04C03 and 1325:01B09. Serum levels of interleukin-6 (IL-6), IL-8, RANKL, OPG and C-terminal crosslinked telopeptide type II collagen were measured by ELISA. Paired-wise Wilcoxon signed-rank test was used to compare median values before and after treatment.Results Disease activity in patients was reduced from baseline to 8 weeks. Although PAD-2 and PAD-4 expressions remained unchanged after TCZ treatment, the binding of one ACPA clone decreased in the synovial tissue. TCZ did not affect the number of CD68+ macrophages or CD20+ B cells but induced significant decrease in the number of CD3+ T cells. RANKL and OPG expression remained unchanged in the synovial tissue. A significant increase in the levels of IL-6 and RANKL was observed in the serum. This increase was statistically significant in patients who responded to TCZ (achieving Clinical Disease Activity Index low disease activity or remission) but not in non-responders.Conclusions TCZ reduced synovial T-cell counts but not macrophages. A significant increase of serum IL-6 was observed in responders

Distinct Fluorescence Optical Imaging Patient Clusters Emerge for Seropositive and Seronegative Rheumatoid Arthritis

Objective To investigate whether digital activity fluorescence optical imaging (FOI) patterns of inflammation can identify distinct rheumatoid arthritis (RA) phenotypes. Methods The hands of newly diagnosed patients with RA were evaluated by clinical examination, musculoskeletal ultrasound, and FOI. Inflammation on FOI was defined when capillary leakage and/or fluorophore perfusion was present. The FOI composite image was quantified into a digital disease activity (DACT) score, using novel computerized algorithms. Unsupervised clustering on FOI inflammatory patterns was used to identify subgroups of patients relative to anticyclic citrullinated peptides (ACPA) and/or rheumatoid factor (RF). Results Of 1326 examined hand joints in 39 patients with RA (72% female; 56% ever‐smokers; 54% RF positive and 69% ACPA positive), 400 (30%) showed inflammation by FOI, and 95% (37 of 39) of patients had DACT‐FOI scores greater than 1. Unsupervised analysis on FOI patterns revealed two patient clusters, cluster 1 (n = 29) and cluster 2 (n = 10). The proportion of seropositive patients was significantly higher in cluster 1 versus cluster 2 (90%, 26 of 29 vs. 30%, 3 of 10; P < 0.01), whereas C‐reactive‐protein levels (minimum‐maximum) were significantly higher in cluster 2 (20 mg/l [1‐102]) versus cluster 1 (2 mg/l [0‐119]; P = 0.01). A wider variety and proportion of inflamed joints emerged for patients with RA in cluster 2 versus cluster 1, in which inflammation was more concentrated around the wrists and the right metacarpophalangeal 2 (MCP2), bilateral MCP3, and, to a lesser degree, left MCP2 and proximal interphalangeal joint and tendon regions. Cluster 1 displayed lower mean (±SD) DACT scores compared with cluster 2 (3.6 ± 2.1 vs. 5.4 ± 2.1; P = 0.03). Conclusion FOI‐based digital quantification of hand joint inflammation revealed two distinct RA subpopulations with and without ACPA and RF related autoantibodies