Synovial membrane immunohistology in early untreated juvenile idiopathic arthritis: differences between clinical subgroups

Abstract: Objective Juvenile idiopathic arthritis (JIA) consists of a heterogeneous group of inflammatory disorders, within which there are a number of clinical subgroups. Diagnosis and assignment to a particular subgroup can be problematical and more concise methods of subgroup classification are required. This study of the synovial membrane characterises the immunohistochemical features in early untreated, newly diagnosed JIA and compares findings with disease subgroup at 2 years.

Methods: 42 patients with newly diagnosed untreated JIA underwent synovial biopsy before the administration of steroids or disease-modifying antirheumatic drugs. Patients were classified as either polyarticular, persistent oligoarticular or extended-to-be oligoarticular. The location and semiquantitative analysis of T-cell subsets, B cells, macrophages and blood vessels were determined using immunohistochemistry.

Results: Synovial hyperplasia varied significantly between the three groups
(p<0.0001). There was a significant difference in the CD3 T-cell population between the three groups (p=0.004) and between the extended-to-be and persistent group (p=0.032). CD4 expression was significantly higher in the poly and extended-to-be oligo groups (p=0.002), again the extended-to-be group had more CD4 T cells than the persistent group (p=0.008). B-cell infiltrates were more marked in the polyarticular group and were significantly higher in the extended-to-be group compared with the persistent group (p=0.005). Vascularisation was more pronounced in the polyarticular and extended-to-be oligoarticular groups, the extended-to-be group had significantly more vascularisation than the persistent group (p=0.0002).

Conclusions: There are significant differences in the histomorphometric features of synovial tissue between patient subgroups. Immunohistological examination of synovial membrane biopsies may provide further insight into early disease processes in JIA.

Synovial membrane protein expression differs between juvenile idiopathic arthritis subtypes in early disease

Introduction: Juvenile idiopathic arthritis (JIA) is the most common rheumatological disease of childhood with a prevalence of around 1 in 1000. Without appropriate treatment it can have devastating consequences including permanent disability from joint destruction and growth deformities. Disease aetiology remains unknown. Investigation of disease pathology at the level of the synovial membrane is required if we want to begin to understand the disease at the molecular and biochemical level. The synovial membrane proteome from early disease-stage, treatment naive JIA patients was compared between polyarticular and oligoarticular subgroups.

Methods: Protein was extracted from 15 newly diagnosed, treatment naive JIA synovial membrane biopsies and separated by two dimensional fluorescent difference in-gel electrophoresis. Proteins displaying a two-fold or greater change in expression levels between the two subgroups were identified by matrix assisted laser desorption ionization-time of flight mass spectrometry with expression further verified by Western blotting and immunohistochemistry.

Results: Analysis of variance analysis (P <= 0.05) revealed 25 protein spots with a two-fold or greater difference in expression levels between polyarticular and oligoarticular patients. Hierarchical cluster analysis with Pearson ranked correlation revealed two distinctive clusters of proteins. Some of the proteins that were differentially expressed included: integrin alpha 2b (P = 0.04); fibrinogen D fragment (P =0.005); collagen type VI (P = 0.03); fibrinogen gamma chain (P = 0.05) and peroxiredoxin 2 (P = 0.02). The identified proteins are involved in a number of different processes including platelet activation and the coagulation system.

Conclusions: The data indicates distinct synovial membrane proteome profiles between JIA subgroups at an early stage in the disease process. The identified proteins also provide insight into differentially perturbed pathways which could influence pathological events at the joint level.

Qualitative study of young, adult, and aged wistar rats temporomandibular synovial membrane employing light, scanning, and transmission electron microscopy

The aim of this study was to analyze the rat temporomandibular joint (TMJ) synovial membrane at different ages using light, scanning, and transmission electron microscopy. Under light microscopic analysis, the TMJ structures were observed such as condyle, capsule, disk, the synovial membrane collagen type, and cells distribution. In the scanning electron microscopy, the synovial membrane surface exhibited a smooth aspect in young animals and there was an increase with ageing in the number of folds. The transmission electron microscopic analysis showed more synoviocytes in the synovial layer in the young group and still a great number of vesicles and cisterns dilation of rough endoplasmic reticulum in the aged group. In the three groups, a dense layer of collagen fibers in the synovial layer and cytoplasmic extensions were clearly seen. It was possible to conclude that synovial membrane structures in aged group showed alterations contributing to the decrease in joint lubrication and in the sliding between disk and joint surfaces. These characteristic will reflect in biomechanics of chewing, and may cause the TMJ disorders, currently observed in clinical processes. Microsc. Res. Tech. (c) 2012 Wiley Periodicals, Inc.

Nitric oxide metabolite production in the cranial cruciate ligament, synovial membrane, and articular cartilage of dogs with cranial cruciate ligament rupture

OBJECTIVE To measure concentrations of nitric oxide metabolites (nitrite-nitrate [NOt]) in cartilage, synovial membrane, and cranial cruciate ligament (CCL) in dogs and evaluate associations with osteoarthritis in dogs with CCL rupture. ANIMALS 46 dogs with CCL rupture and 54 control dogs without joint disease. PROCEDURE Tissue specimens for histologic examination and explant culture were harvested during surgery in the CCL group or immediately after euthanasia in the control group; NOt concentrations were measured in supernatant of explant cultures and compared among dogs with various degrees of osteoarthritis and between dogs with and without CCL rupture. RESULTS Osteoarthritic cartilage had significantly higher NOt concentration (1,171.6 nmol/g) than did healthy cartilage (491.0 nmol/g); NOt concentration was associated with severity of macroscopic and microscopic lesions. Synovial membrane NOt concentration did not differ between dogs with and without CCL rupture. Ruptured CCL produced less NOt than did intact ligaments. In control dogs, NOt concentrations were similar for intact ligaments (568.1 nmol/g) and articular cartilage (491.0 nmol/g). Synthesis of NOt was inhibited substantially by coincubation with inhibitors. CONCLUSIONS AND CLINICAL RELEVANCE Results suggest that NOt in canine joint tissues originates from the inducible nitric oxide synthase pathway. Nitric oxide metabolite production in cartilage was greater in dogs with osteoarthritis than in healthy dogs and was associated with lesion severity, suggesting that nitric oxide inhibitors may be considered as a treatment for osteoarthritis. The CCL produces substantial concentrations of NOt; the importance of this finding is unknown.

B lymphocyte involvement in ankylosing spondylitis: the heavy chain variable segment gene repertoire of B lymphocytes from germinal center-like foci in the synovial membrane indicates antigen selection

The synovial membrane (SM) of affected joints in ankylosing spondylitis (AS) is infiltrated by germinal center-like aggregates (foci) of lymphocytes similar to rheumatoid arthritis (RA). We characterized the rearranged heavy chain variable segment (VH) genes in the SM for gene usage and the mutational pattern to elucidate the B lymphocyte involvement in AS.

Differentiation of B cells in the nonlymphoid tissue of the synovial membrane of patients with rheumatoid arthritis.

In patients with rheumatoid arthritis the synovial membrane of the affected joint is infiltrated with lymphoid cells which may be arranged in structures resembling germinal centers. We have directly isolated such infiltrates to determine whether B-cell clones within them are selected and expanded in a process analogous to that which normally takes place in the germinal centers in secondary lymphoid organs. The data suggest that an antigen-driven process leads to the accumulation of B cells in the synovial membrane. The finding of identical sequences in consecutive sections suggests that under conditions of chronic stimulation, memory B cells may enter a stage of differentiation in which they proliferate without further accumulation of somatic mutations. Further we see intraclonal diversity which underlines the germinal center-like character of these infiltrates and demonstrates that a microenvironment is built up in this nonlymphoid tissue which supports antigen-dependent differentiation of B cells. This is the first demonstration, to our knowledge, of a germinal center-like reaction outside lymphoid tissue.

Expression profiling in spondyloarthropathy synovial biopsies highlights changes in expression of inflammatory genes in conjunction with tissue remodelling genes

Background: In the spondyloarthropathies, the underlying molecular and cellular pathways driving disease are poorly understood. By undertaking a study in knee synovial biopsies from spondyloarthropathy (SpA) and ankylosing spondylitis (AS) patients we aimed to elucidate dysregulated genes and pathways. Methods RNA was extracted from six SpA, two AS, three osteoarthritis (OA) and four normal control knee synovial biopsies. Whole genome expression profiling was undertaken using the Illumina DASL system, which assays 24000 cDNA probes. Differentially expressed candidate genes were then validated using quantitative PCR and immunohistochemistry. Results: Four hundred and sixteen differentially expressed genes were identified that clearly delineated between AS/SpA and control groups. Pathway analysis showed altered gene-expression in oxidoreductase activity, B-cell associated, matrix catabolic, and metabolic pathways. Altered «myogene» profiling was also identified. The inflammatory mediator, MMP3, was strongly upregulated (5-fold) in AS/SpA samples and the Wnt pathway inhibitors DKK3 (2.7-fold) and Kremen1 (1.5-fold) were downregulated. Conclusions: Altered expression profiling in SpA and AS samples demonstrates that disease pathogenesis is associated with both systemic inflammation as well as local tissue alterations that may underlie tissue damaging modelling and remodelling outcomes. This supports the hypothesis that initial systemic inflammation in spondyloarthropathies transfers to and persists in the local joint environment, and might subsequently mediate changes in genes directly involved in the destructive tissue remodelling.

Expression of protease-activated receptors in arthritic synovial tissues

Clinical and experimental evidence suggests that synovial thrombin formation in arthritic joints is prominent and deleterious, leading to exacerbation of rheumatoid arthritis (RA). In this context, cellular effects of thrombin mediated by the protease-activated receptors (PARs) in arthritic joints may be of paramount significance. Four PARs have now been identified. PAR1, PAR3, and PAR4 can all be activated by thrombin whereas PAR2 is activated by trypsin and few other proteases.We first explored PARs expression in RA synovial tissues. Synovial membranes from 11 RA patients were analyzed for PARs expression by RT-PCR and by immunohistology. PAR4 was found in all the biopsies, whereas the expression of PAR1, PAR 2 and PAR3 was more restricted (8/11, 5/11 and 3/11 respectively). In the arthritic synovial membrane of murine antigen-induced arthritis (AIA) we found coexpression of the four different PARs. Next, we explored the functional importance of PAR1 during AIA in vivo using PAR-1 deficient mice. The phenotype of PAR1-deficient mice (n = 22), based on the analysis of arthritis severity (as measured by 99 m tecnetium uptake, histological scoring and intra-articular fibrin measurements) was similar to that of wild-type mice (n = 24). In addition, the in vivo production of antibodies against mBSA was also similar. By contrast, the mBSA-induced in vitro lymph node cell proliferation was significantly decreased in PAR1-deficient mice as compared with controls. Accordingly, mBSA-induced production of interferon-γ by lymph node cells in culture was significantly decreased in PAR1-deficient mice as compared with controls, whereas opposite results were observed for production of IL-10.

Modified complete synovectomy prevents recurrence in synovial chondromatosis of the hip

Synovial chondromatosis of the hip is a rare disorder with few published reports regarding treatment and outcomes, and therefore, selecting the optimal surgical treatment is difficult. We reviewed eight patients with monoarticular synovial chondromatosis of the hip who had joint débridement and a modified total synovectomy performed through a surgical hip dislocation with a trochanteric flip osteotomy. Patients were evaluated for recurrence of disease, progression of osteoarthritis, clinical outcomes, and subsequent reoperations. The minimum followup was 4 years (mean, 6.5 years). At final review, no patient had recurrence of disease. Two patients had progression of osteoarthritis requiring total hip arthroplasties at 5 and 10 years after the initial surgical intervention. These patients did not show recurrent disease on histologic examination of the synovial membrane at the time of the arthroplasty. The six patients with preserved joints were followed up for a mean of 6.2 years. The mean Merle d'Aubigné and Postel score in this group was 16.5 points (range, 15-18 points) at the latest followup. There were no major or minor complications related to this treatment. Our midterm results suggest that open débridement with modified total synovectomy is an effective treatment that prevents recurrence of disease and provides substantial pain relief. Surgical hip dislocation allows safe and complete access to the joint for débridement and synovectomy with no added morbidity.

Endogenous nitric oxide production in canine osteoarthritis: Detection in urine, serum, and synovial fluid specimens

OBJECTIVE To measure nitric oxide (NO) concentrations in serum, urine, and synovial fluid (SF) of dogs with naturally occurring cranial cruciate ligament (CCL) rupture and normal dogs, and to compare these with clinical and histologic changes of osteoarthritis (OA). STUDY DESIGN Prospective clinical study including 2 groups of animals selected from the hospital population. ANIMALS Forty-three dogs (CCL group) with OA secondary to CCL rupture; 30 healthy dogs (control group) without CCL rupture. METHODS Serum, urine, and SF were collected before and during surgery in the CCL group or immediately after euthanasia in the control group. Articular cartilage and synovial membrane tissue specimens were prepared for routine histologic examination. The stable end products of NO, total nitrite and nitrate (NOt) activity, were measured in body fluids and compared with macroscopic and histologic degrees of OA. Urinary NOt concentration was compared with urinary creatinine concentration and stated as urinary NOt:creatinine ratio (UNCR). RESULTS-SF NOt concentrations were not significantly different between the 2 groups. Serum NOt concentrations (45.6 vs 28.9 micromol/L; P =.042) and the UNCR (0.007 vs 0.004; P =.035) were significantly higher in dogs of the CCL group compared with the control population. An association between UNCR and histologic and macroscopical OA grades could be demonstrated. CONCLUSION UNCR might be a useful indicator of nitrite and nitrate production and, therefore, osteoarthritic changes in joints. CLINICAL RELEVANCE UNCR could be used as a tool to evaluate the NOt production by joint tissues over time and might therefore provide a method of evaluating the effects of drugs in the control of osteoarthritis.

Fine mapping of the MHC Class III region demonstrates association of AIF1 and rheumatoid arthritis

Objective. The heritability of RA has been estimated to be ∼55%, of which the MHC contributes about one-third. HLA-DRB1 alleles are strongly associated with RA, but it is likely that significant non-DRB1 MHC genetic susceptibility factors are involved. Previously, we identified two three-marker haplotypes in a 106-kb region in the MHC class III region immediately centromeric to TNF, which are strongly associated with RA on HLA-DRB1*0404 haplotypes. In the present study, we aimed to refine these associations further using a combination of genotyping and gene expression studies. Methods. Thirty-nine nucleotide polymorphisms (SNPs) were genotyped in 95 DRB1*0404 carrying unrelated RA cases, 125 DRB1*0404 - carrying healthy controls and 87 parent-case trio RA families in which the affected child carried HLA-DRB1*04. Quantitative RT-PCR was used to assess the expression of the positional candidate MHC class III genes APOM, BAT2, BAT3, BAT4, BAT5, AIF1, C6orf47, CSNK2β and LY6G5C, and the housekeeper genes, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and β2-microglobulin (B2M) in 31 RA cases and 21 ethnically, age- and sex-matched healthy controls. Synovial membrane specimens from RA, PsA and OA cases were stained by an indirect immunoperoxidase technique using a mouse-anti-human AIF1 monoclonal antibody. Results. Association was observed between RA and single markers or two marker haplotypes involving AIF1, BAT3 and CSNK. AIF1 was also significantly overexpressed in RA mononuclear cells (1.5- to 1.9-fold difference, P = 0.02 vs HPRT, P = 0.002 vs B2M). AIF1 protein was clearly expressed by synovial macrophages in all the inflammatory synovial samples in contrast to the non-inflammatory OA samples. Conclusions. The results of the genotyping and expression studies presented here suggest a role for AIF1 in both the aetiology and pathogenesis of RA.

ADAMs, Osteoclastogenesis and Bone Destruction in the Loosening of the Totally Replaced Hip

Total hip replacement is the golden standard treatment for severe osteoarthritis refractory for conservative treatment. Aseptic loosening and osteolysis are the major long-term complications after total hip replacement. Foreign body giant cells and osteoclasts are locally formed around aseptically loosening implants from precursor cells by cell fusion. When the foreign body response is fully developed, it mediates inflammatory and destructive host responses, such as collagen degradation. In the present study, it was hypothesized that the wear debris and foreign body inflammation are the forces driving local osteoclast formation, peri-implant bone resorption and enhanced tissue remodeling. Therefore the object was to characterize the eventual expression and the role of fusion molecules, ADAMs (an abbreviation for A Disintegrin And Metalloproteinase, ADAM9 and ADAM12) in the fusion of progenitor cells into multinuclear giant cells. For generation of such cells, activated macrophages trying to respond to foreign debris play an important role. Matured osteoclasts together with activated macrophages mediate bone destruction by secreting protons and proteinases, including matrix metalloproteinases (MMPs) and cathepsin K. Thus this study also assessed collagen degradation and its relationship to some of the key collagenolytic proteinases in the aggressive synovial membrane-like interface tissue around aseptically loosened hip replacement implants. ADAMs were found in the interface tissues of revision total hip replacement patients. Increased expression of ADAMs at both transcriptional and translational levels was found in synovial membrane-like interface tissue of revision total hip replacement (THR) samples compared with that in primary THR samples. These studies also demonstrate that multinucleate cell formation from monocytes by stimulation with macrophage-colony stimiulating factor (M-CSF) and receptor activator of nuclear factor kappa B ligand (RANKL) is characterized by time dependent changes of the proportion of ADAMs positive cells. This was observed both in the interface membrane in patients and in two different in vitro models. In addition to an already established MCS-F and RANKL driven model, a new virally (parainfluenza 2) driven model (of human salivary adenocarcinoma (HSY) cells or green monkey kidney (GMK) cells) was developed to study various fusion molecules and their role in cell fusion in general. In interface membranes, collagen was highly degraded and collagen degradation significantly correlated with the number of local cells containing collagenolytic enzymes, particularly cathepsin K. As a conclusion, fusion molecules ADAM9 and ADAM12 seem to be dynamically involved in cell-cell fusion processes and multinucleate cell formation. The highly significant correlation between collagen degradation and collagenolytic enzymes, particularly cathepsin K, indicates that the local acidity of the interface membrane in the pathologic bone and soft tissue destruction. This study provides profound knowledge about cell fusion and mechanism responsible for aseptic loosening as well as increases knowledge helpful for prevention and treatment.

Pannus invasion into cartilage and bone in rheumatoid arthritis

Rheumatoid arthritis is the most common of all types of arthritis and despite of intensive research etiology of the disease remains unclear. Distinctive features of rheumatic arthritis comprise continuous inflammation of synovium, in which synovial membrane expands on cartilage leading to pannus tissue formation. Pannus formation, appearance of proteolytic enzymes and osteoclast formation cause articular cartilage and bone destruction, which lead to erosions and permanent joint damage. Proteolytic pathways play major roles in the development of tissue lesions in rheumatoid arthritis. Degradation of extracellular matrix proteins is essential to pannus formation and invasion. Matrix metalloproteinases (MMP) form a large proteolytic enzyme family and in rheumatoid arthritis they contribute to pannus invasion by degrading extracellular matrix and to joint destruction by directly degrading the cartilage. MMP-1 and MMP-3 are shown to be increased during cell invasion and also involved in cartilage destruction. Increase of many cytokines has been observed in rheumatoid arthritis, especially TNF-α and IL-1β are studied in synovial tissue and are involved in rheumatoid inflammation and degradation of cartilage. Underlying bone resorption requires first demineralization of bone matrix with acid secreted by osteoclasts, which exposes the collagen-rich matrix for degradation. Cathepsin K is the best known enzyme involved in bone matrix degradation, however deficiency of this protein in pycnodysostosis patient did not prevent bone erosion and on the contrary pannus tissue invading to bone did not expressed much cathepsin K. These indicate that other proteinases are involved in bone degradation, perhaps also via their capability to replace the role of other enzymes especially in diseases like pycnodysostosis or during medication e.g. using cathepsin K inhibitors. Multinuclear osteoclasts are formed also in pannus tissue, which enable the invasion into underlying bone matrix. Pannus tissue express a receptor activator of nuclear factor kappa B ligand (RANKL), an essential factor for osteoclast differentiation and a disintegrin and a metalloproteinase 8 (ADAM8), an osteoclast-activating factors, involved in formation of osteoclast-like giant cells by promoting fusion of mononuclear precursor cells. The understanding of pannus invasion and degradation of extracellular matrix in rheumatic arthritis will open us new more specific methods to prevent this destructive joint disease.

A new approach to depict bone surfaces in finger imaging using photoacoustic tomography

Imaging the vasculature close around the finger joints is of interest in the field of rheumatology. Locally increased vasculature in the synovial membrane of these joints can be a marker for rheumatoid arthritis. In previous work we showed that part of the photoacoustically induced ultrasound from the epidermis reflects on the bone surface within the finger. These reflected signals could be wrongly interpreted as new photoacoustic sources. In this work we show that a conventional ultrasound reconstruction algorithm, that considers the skin as a collection of ultrasound transmitters and the PA tomography probe as the detector array, can be used to delineate bone surfaces of a finger. This can in the future assist in the localization of the joint gaps. This can provide us with a landmark to localize the region of the inflamed synovial membrane. We test the approach on finger mimicking phantoms.

Photoacoustic tomography of the human finger: towards the assessment of inflammatory joint diseases

Inflammatory arthritis is often manifested in finger joints. The growth of new or withdrawal of old blood vessels can be a sensitive marker for these diseases. Photoacoustic (PA) imaging has great potential in this respect since it allows the sensitive and highly resolved visualization of blood. We systematically investigated PA imaging of finger vasculature in healthy volunteers using a newly developed PA tomographic system. We present the PA results which show excellent detail of the vasculature. Vessels with diameters ranging between 100 mu m and 1.5 mm are visible along with details of the skin, including the epidermis and the subpapillary plexus. The focus of all the studies is at the proximal and distal interphalangeal joints, and in the context of ultimately visualizing the inflamed synovial membrane in patients. This work is important in laying the foundation for detailed research into PA imaging of the phalangeal vasculature in patients suffering from rheumatoid arthritis.