Variability of RANKL and osteoprotegerin staining in synovial tissue from patients with active rheumatoid arthritis: quantification using color video image analysis

OBJECTIVE: To assess the interpatient, interbiopsy, and intrabiopsy variability of receptor activator of nuclear factor kB ligand (RANKL) and osteoprotegerin (OPG) immunostaining within synovial tissue from rheumatoid knee joints with active synovitis, using digital image analysis. METHODS: Synovial biopsy specimens were obtained from patients with rheumatoid arthritis (RA) and active synovitis. Immunohistologic analysis was performed on frozen synovial tissue biopsy specimens from 6 patients using a monoclonal antibody (Mab) to detect RANKL (626) or OPG (805 or 8051). Patients with a minimum of 4 synovial biopsies were included in the study. Sections were evaluated by computer assisted image analysis to assess between-patient, between-biopsy, and intra-biopsy variability of OPG and RANKL protein expression. The study was designed to deliberately maximize the variability. RESULTS: Computerized image analysis of staining with Mab to RANKL and OPG revealed variance for each antibody across the 3 components of the total variability. CONCLUSION: Our study shows that variability in synovial immunostaining of RANKL and OPG protein is a significant and complex problem. We discuss methods to reduce this variability and suggest that the auspices of OMERACT may be employed to advance the study of synovium in collaborative international studies.     

Morphometric analysis of blood vessels in synovial membranes obtained from clinically affected and unaffected knee joints of patients with rheumatoid arthritis.

Synovial tissues from inflamed and noninflamed knee joints of 13 patients with untreated rheumatoid arthritis were examined for vascular proliferation and morphological alteration of endothelial cells. Perivascular mononuclear cell infiltration and increased thickness of the synovial lining layer were noted in tissues from inflamed and noninflamed joints of patients with rheumatoid arthritis; vascular proliferation and morphological alteration of endothelial cells to resemble high endothelial venules were seen only in tissues from inflamed joints of patients with rheumatoid arthritis. These observations suggest that the migration of mononuclear cells from the peripheral blood to the perivascular areas and lining layer occurs before vascular proliferation and morphological alteration of endothelial cells.     

Protoporphyrin IX photodynamic therapy for synovitis

OBJECTIVE: To determine the conditions for synovial accumulation of protoporphyrin IX (PpIX) and photodynamic therapy (PDT)-induced synovial cytotoxicity in vitro and in vivo. METHODS: Synovial tissues were obtained from mice with antigen-induced arthritis (AIA) and incubated with different concentrations of 5-aminolevulinic acid hexyl ester (h-ALA), a PpIX precursor. Following photoexcitation, cell death in synovial tissues was evaluated by Sytox green fluorescence. PDT was performed after intraarticular injection of h-ALA into the knee joints of mice with AIA, and its effect on joint inflammation was assessed by technetium scintigraphy and histology. Synovial biopsy samples were obtained from patients with osteoarthritis (OA; n = 9) and rheumatoid arthritis (RA; n = 7) and studied for PDT-induced cytotoxicity in vitro. RESULTS: Conversion of h-ALA to PpIX was observed in inflamed synovium in mice and humans. Cytotoxicity was confirmed by Sytox green staining in samples subjected to PDT. In the AIA model, injection of affected knees with h-ALA prior to PDT led to a statistically significant reduction of joint damage in the irradiated joints. The preferential transformation of h-ALA to PpIX in inflammatory tissues was confirmed in human synovial biopsy tissues, where RA samples showed higher tissue concentrations of PpIX following incubation with h-ALA than did OA samples. Fluorescence microscopy showed that PpIX was localized to the synovial lining layer, endothelial cells, and macrophages and induced cell death after PDT. CONCLUSION: Our findings suggest that PDT based on the accumulation of PpIX in the synovial membrane may be a rational basis for photodynamic synovectomy in arthritic diseases.     

Immunohistological analysis of the synovial membrane: search for predictors of the clinical course in rheumatoid arthritis.

Immunohistological features which might predict the clinical course and outcome of rheumatoid arthritis were sought by examining multiple synovial membrane samples obtained by needle biopsy from the knee joints of 57 patients who had not received disease modifying antirheumatic drugs. Clinical measurements, but not biopsies, were repeated one year and three years after starting treatment. A correlation between both the intensity of synovial lining layer thickening and mononuclear cell infiltration and the clinical status at the time of biopsy was seen. After three years of treatment the correlations were maintained in patients who had presented and persisted with milder disease but not in patients who had presented with more active disease.     

Presence of foam cells containing oxidised low density lipoprotein in the synovial membrane from patients with rheumatoid arthritis.

OBJECTIVE--Increased concentrations of lipid peroxidation products have been described in the serum and synovial fluid from patients with rheumatoid arthritis. A large proportion of the unsaturated lipids in human extracellular fluids is a component of low density lipoprotein (LDL). The oxidative modification of LDL, and its subsequent uptake by macrophages, has been implicated in the pathogenesis of atherosclerosis, but not of rheumatoid arthritis. This study aimed to assess whether oxidatively modified LDL was present in the rheumatoid synovium. METHODS--A polyclonal antiserum raised in rabbits against oxidised LDL (o-LDL) was used to perform an immunohistochemical study of a series of synovial biopsy specimens from patients with rheumatoid arthritis. RESULTS--Collections of positively stained macrophages, arranged in a linear fashion and with the morphological characteristics of foam cells--that is, 'fatty streaks', were identified around blood vessels within the intimal connective tissue. In addition, scattered, positively stained foam cells were present in association with deposits of fibrin. These staining patterns were absent from control synovial membranes (traumatic knee injuries). CONCLUSIONS--The findings in all rheumatoid patients studied suggest that atherosclerosis and rheumatoid arthritis have analogous pathogenetic features.     

Neuroendocrine-immune interactions in synovitis

Synovial tissue lines the noncartilaginous surfaces of synovial joints and supplies these avascular structures with nutrients. In diseases such as rheumatoid arthritis, inflammation of the synovial tissue--synovitis--induces diffuse damage to the joints. The presence of functional receptors for glucocorticoids, androgens and estrogens in synoviocytes might link inflammation and the endocrine system at the local level. Synovial tissue could be regarded as an intracrine tissue, whereby active steroids influence the cells in which they are synthesized, without their release into the extracellular space. An increase in the peripheral metabolism of sex steroids is characteristic of rheumatoid synovitis, with an augmented ratio of estrogen to androgen occurring in both male and female patients. Changes in the peripheral nervous system at the site of local inflammation are also hallmarks of synovitis in rheumatoid arthritis. In the chronic phase of synovitis, sympathetic nerve fibers are lost; by contrast, sensory nerve fibers sprout into the inflamed tissue. Complex interactions occur between the endocrine, nervous and immune systems during synovitis. In particular, studying neuroendocrine-immune interactions in the inflamed synovium will potentially uncover new mechanisms in the pathophysiology of rheumatoid arthritis and might lead to new methods of therapeutic intervention.     

Differences in synovial tissue infiltrates between anti-cyclic citrullinated peptide-positive rheumatoid arthritis and anti-cyclic citrullinated peptide-negative rheumatoid arthritis.

OBJECTIVE: To compare synovial tissue infiltrates from patients with anti-cyclic citrullinated peptide (anti-CCP)-positive rheumatoid arthritis (RA) with those from patients with anti-CCP-negative RA. METHODS: Synovial tissue samples were obtained arthroscopically from the inflamed knee joints of 57 patients with RA (34 of whom were anti-CCP positive) and examined for several histologic features along with immunohistologic expression of cell markers. Joint damage was assessed using the Kellgren/Lawrence (K/L) scale (range 0-4) on standard anteroposterior radiographs. In 31 patients (18 of whom were anti-CCP positive), synovial tissue was available from an earlier time point, allowing analysis of temporal changes. RESULTS: Synovial tissue from anti-CCP-positive patients was characterized by a higher mean number of infiltrating lymphocytes (61.6 versus 31.4/high-power field [hpf] [400x]; P=0.01), less extensive fibrosis (mean score of 1.2 versus 2.0; P=0.04), and a thinner synovial lining layer (mean score of 2.1 versus 3.3; P=0.002) compared with synovial tissue from anti-CCP-negative patients. Anti-CCP-positive patients expressed more CD3, CD8, CD45RO, and CXCL12. More anti-CCP-positive patients had a K/L score >1 compared with anti-CCP-negative patients. The difference in the mean lymphocyte counts was already present a mean of 3.8 years before the index biopsy (76.7 lymphocytes/hpf and 26.7 lymphocytes/hpf in anti-CCP-positive patients and anti-CCP-negative patients, respectively; P=0.008) and was independent of disease duration and K/L score. CONCLUSION: Synovitis in patients with anti-CCP-positive RA differs from that in patients with anti-CCP- negative RA, notably with respect to infiltrating lymphocytes, and is associated with a higher rate of local joint destruction.     

Morphology and growth behavior of synovial cells in monolayer culture

Synovial fluid cells from patients with rheumatoid arthritis, psoriatic arthritis, peripheral arthritis in ankylosing spondylitis, dialysis arthropathy, osteoarthrosis, and joint disorders due to acute trauma were grown in monolayer cultures and examined by light and electron microscopy at arbitrarily chosen times and in various subcultures. The cultivated cells from these sources were compared with the cells of synovial tissue (cultured under the same conditions) from patients with osteoarthrosis and traumatized joints. Our results are not in agreement with those of most other authors. In contrast to many authors, we could not find any differences between the cultures from synovial fluid and the cultures from synovial tissue: the same cell types were observed with a slight quantitative difference in both cultures. Furthermore, no morphological features (except an abundance of filopodia of rheumatoid polykaryocytes) which would be characteristic of the rheumatoid synovial cells could be determined. Due to our experiments the synovial fluid seems to be more advantageous than synovial tissue for cell culture studies of different types of arthritis. This may be attributed to the following factors: a) the easy accessibility of synovial-fluid samples by ambulant aspiration from affected joints; b) the possibility of repeating this procedure frequently without any essential danger for the patient; c) the maintainability of synovial fluid (when chilled to 4 degrees C) up to 2-4 days without cessation of the growth ability of the cells under normal culture conditions. Apparently, synovial fluid represents an appropriate medium for synovial cells to survive in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Citrullination is an inflammation-dependent process

OBJECTIVES: To study the presence of citrullinated proteins in inflammatory conditions and in clinically non-affected tissues of controls. METHODS: Synovial biopsy specimens from 19 patients with rheumatoid arthritis and 10 healthy controls were investigated by immunohistochemistry. Additionally, muscle tissue from 5 patients with polymyositis and from 7 healthy controls, intestinal tissue from macroscopically affected and non-affected areas from 10 patients with inflammatory bowel disease (IBD) and tonsil tissues from 4 chronically inflamed tonsils were studied. RESULTS: Citrullinated proteins were present in all synovial biopsy specimens from patients with rheumatoid arthritis, whereas only three of 10 healthy synovial biopsy specimens showed scarce amounts of citrullination. Citrullination was also present in all myositis-affected muscles, whereas it was absent in the muscle tissues of controls. All tonsil biopsy specimens studied were positive for citrulline. Even though more frequently detected in the macroscopically affected colonic areas, no marked difference was observed in the pattern or extent of citrullination between the macroscopically affected and non-affected intestinal IBD tissues. CONCLUSION: Citrullination is present in a wide range of inflammatory tissues, suggesting that this process is inflammation dependent rather than disease dependent.     

Diagnostic value of blind synovial biopsy in clinical practice

OBJECTIVE: To assess the diagnostic value of blindly performed synovial biopsies in carefully selected patients with unclassified arthritis. METHODS: Synovial tissue was obtained blindly under local anaesthesia. The Arthroforce III take-apart 3.5 mm needle and 1.5 mm grasping forceps were used for this purpose. RESULTS: Four patients with unclassified arthritis could be diagnosed properly based upon examination of synovial tissue of the knee obtained by an easy-to-perform blind biopsy. The arthritis of the four patients was diagnosed as being part of Erdheim-Chester disease, sarcoidosis, multicentric reticulohistiocytosis and arthritis caused by foreign-body material, respectively. CONCLUSIONS: Analysis of synovial tissue obtained during a blind biopsy procedure has diagnostic potential in carefully selected patients with unclassified arthritis. The common denominator in all the cases presented was a differential diagnosis consisting of a rheumatological disease with characteristic histological features.     

THE DIAGNOSTIC VALUE OF SYNOVIA BIOPSY IN PATIENTS WITH ARTHRITIS OF UNKNOWN CAUSE

Synovial biopsy of a knee joint was performed in 59 patientswith arthritis of unknown cause. Light microscopy revealed adiagnosis in three. A cause became apparent in only a further23 over a prolonged period of follow-up. Neither light microscopy,immunofluorescence nor electron microscopy discriminated betweenthe principal diagnostic groups. Synovial fluid analysis wasbetter than synovial biopsy at predicting osteoarthritis. Fivemen with giant effusions developed osteoarthritis and thesemight represent an entity. Synovial inflammatory changes wereless pronounced in those with longer histories. Disease durationand activity may have more influence on synovial characteristicsthan the nature of the disorder. KEY WORDS: Diagnosis, Synovium, Light microscopy, Immunofluorescence, Electron microscopy     

Interleukin-6 levels in synovial fluids of patients with rheumatoid arthritis correlated with the infiltration of inflammatory cells in synovial membrane

To compare the histological appearance of synovial membrane and interleukin (IL)-6 levels in synovial fluids of patients with rheumatoid arthritis (RA). Synovial tissue and synovial fluids were obtained from 51 knee joints with RA undergoing synovectomy or joint replacements. A histological inflammation score was determined based on the hyperplasia of the synovial lining and infiltration of inflammatory cells. The concentrations of IL-6 in synovial fluids were measured by ELISA. The association between IL-6 levels and histological findings was evaluated. We found a positive correlation between the infiltration of inflammation cells in synovial tissues and the concentration of IL-6 in synovial fluids. The IL-6 level in synovial fluid partially reflects histological synovial inflammation.     

Amyloid arthritis simulating rheumatoid disease in five patients with multiple myeloma

Five patients with multiple myeloma, three of whom had kappa light chain disease, presented with a symmetrical polyarthritis simulating rheumatoid arthritis. In all instances amyloid infiltration of synovial tissues appeared to account for the articular manifestations. Synovial fluids examined in four of these cases lacked the inflammatory characteristics of rheumatoid synovial fluid and three of them contained M components. In the course of synovial fluid analysis, small fragments of amyloid-containing material were detected in the aspirates from three of the patients, thereby establishing the diagnosis prior to confirmation by tissue biopsy. In the past patients with verified amyloidosis involving synovial tissues have been erroneously considered to have rheumatoid arthritis.     

Synovial lipoma arborescens

Synovial lipoma arborescens is a rare and benign intra-articular pathology, of unknown etiology, characterized by a villous and lipomatous proliferation of synovial tissue. It presents with atypical clinical manifestations, usually located in the knee, represented as recurrent joint effusions and painless swelling joint. The magnetic resonance is the most specific test and can often even avoid the synovial biopsy. We related the case of a female patient with mechanical pain in the knee with indolent evolution for 18 years, clinical and radiological compatible with osteoarthritis. With the finding of a localized unilateral increase of the suprapatellar bursa without perceptible joint effusion and ultrasonographic aspect of an exuberant nodular synovitis, the possibility of villonodular pigmented synovitis had to be discarded by synovial biopsy. Even after this procedure, her diagnosis was not clear, being reported to rheumatology evaluation due to histopathology findings confused with rheumatoid arthritis. The set of clinical, laboratory, magnetic resonance and histological review of synovial tissue confirmed the diagnosis of synovial lipoma arborescens, excluding the possibility of rheumatoid arthritis.     

An investigation of the action of disease modifying antirheumatic drugs on the rheumatoid synovial membrane: reduction in T lymphocyte subpopulations and HLA-DP and DQ antigen expression after gold or penicillamine therapy.

Synovial needle biopsy specimens from the knee joints of seven patients with rheumatoid arthritis (RA) were examined immunohistochemically before and after six months' treatment with either gold or penicillamine (disease modifying drugs, DMDs). There were significant reductions in the numbers of infiltrating T lymphocytes and a disproportionate fall in the numbers of lymphocytes of the helper/inducer subset when compared with those of the suppressor/cytotoxic subset. This resulted in a fall in the ratio of helper/inducer to suppressor/cytotoxic cells. The immunohistological changes correlated with improvements in erythrocyte sedimentation rate (ESR), serum immunoglobulins, visual analogue pain assessment, grip strength, and Ritchie articular index. A second group of nine patients with RA, already well established on DMD therapy, did not show similar changes after the six month period. The HLA class II antigens DR, DQ, and DP were widely expressed on lymphocytes, macrophages, and synovial lining cells of a group of patients with RA who had never received disease modifying drug therapy. After treatment there was a significant reduction in the expression of HLA-DP and DQ antigens.     

Analysis of the histologic variation of synovitis in rheumatoid arthritis

One hundred forty-five synovial biopsy specimens were obtained from 30 procedures performed on the knee joints of 29 patients with rheumatoid arthritis. All patients had clinically active rheumatoid arthritis and none had received slow-acting disease-modifying drugs or intraarticular corticosteroids. Scores were assigned to each biopsy specimen for each of 6 histologic features to quantify variation within each joint. In the majority of knee joint biopsies, there was considerable clustering of scores for all histologic features. Thus, on a scale of 0-10, 82% of the scores for synoviocyte hyperplasia were within 1 point of the median score for a given joint. Similarly, between 69% and 85% of the scores for the remaining features (fibrosis, vessel proliferation, perivascular infiltrates, focal aggregates, and diffuse infiltrates of lymphocytes) were within 1 point of the median values. Multiple biopsies were obtained at arthroscopy in 8 patients. Tissue was selected from areas of apparent maximal and minimal involvement, to enhance the likelihood of regional histologic variation. Of the scores for synoviocyte hyperplasia, 91% were within 1 point of the median values for a given joint, and of the scores for the remaining 5 features, 72-94% fell within 1 point of the median values. In addition, highly significant statistical correlations of the intensity of synovial lining layer hyperplasia, vessel proliferation, mononuclear cell infiltration, fibrosis, and clinical measurements of synovitis were observed.     

Chlamydial rRNA in the joints of patients with Chlamydia-induced arthritis and undifferentiated arthritis.

Synovial fluid and synovial membrane specimens of 11 patients with Chlamydia-induced arthritis (CIA), 24 patients with undifferentiated arthritis (UndA), 4 patients with post-enteritic reactive arthritis, 3 patients with Lyme arthritis and 9 patients with rheumatoid arthritis were investigated for the presence of Chlamydia trachomatis (C. trachomatis). A single stranded DNA-probe was used for nucleic acid hybridization with ribosomal RNA (rRNA) from C. trachomatis. In 4 patients (CIA = 1, UndA = 3) chlamydial rRNA was found in the synovial fluid. In one additional patient (CIA) the specimen of a synovial membrane biopsy was positive for chlamydial rRNA. The detection of intra-articular chlamydial rRNA is discussed as an indicator for the presence of viable Chlamydiae in inflamed joints.     

Mode of formation of synovial villi.

Synovial tissue from 6 normal pigs and from 16 patients undergoing arthrotomy for joint disease was examined by dissecting microscopy. Scale models were constructed of 3 human synovial specimens from photographic magnifications of serial sections. Surface bridging and subintimal cavitation were observed, particularly in tissue from patients with rheumatoid arthritis. These features suggest that synovial surface projections (villi) do not form simply by outgrowth. Reference to original haematoxylin and eosin stained sections suggested that tissue splitting contributes to the formation of villi.     

SYNOVIAL FLUIDS FROM INFECTED JOINTS CONTAIN ACTIVE METALLOPROTEINASES AND NO INHIBITORY ACTIVITY

Serial samples of synovial fluid aspirated from two patientswith septic arthritis were assayed for pro-teinases and proteinaseinhibitors. Active metalloproteinases but no proteinase inhibitorswere present in all samples taken prior to treatment. The levelsof active metalloproteinases fell with time although proenzymeforms were still present in the fluids. Both or2-macroglobulinand the tissue inhibitor of metalloproteinases were found inthe septic synovial fluids after treatment commenced. It isproposed that the lack of inhibitors and the presence of activeproteinases capable of digesting collagen, gelatin and proteoglycanaccounts for the rapid loss of cartilage found in septic arthritis. KEY WORDS: Collagenase, T1MP, Synovial fluid, Macroglobulin, Gelatinase, Septic arthritis