Plasminogen activators and their inhibitors in synovial fluids from normal, osteoarthritis, and rheumatoid arthritis knees.

OBJECTIVES: To establish baseline concentrations of plasminogen activators and their inhibitors in normal knee synovial fluids, and to compare them with well characterised osteoarthritis (OA) and rheumatoid arthritis (RA) knee fluids. METHODS: A total of 26 normal subjects, 71 patients with OA, and 17 patients with RA underwent knee aspiration. Patients with OA were subclassified according to presence of nodal generalised OA (NGOA) and synovial fluid calcium pyrophosphate crystals. Clinical assessment of inflammation (graded 0-6) was undertaken in OA and RA patients. Plasminogen activator (PA), plasminogen activator inhibitor (PAI), and urokinase-type PA receptor (uPAR) antigen concentrations were determined by enzyme linked immunosorbent assay. The species of PAs present were determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. RESULTS: Concentrations of all antigens (uPA, tissue-type PA (tPA), uPAR, and PAI-1), were significantly greater in RA than OA; those in OA were significantly greater than normal. The concentrations showed no direct association with clinically assessed inflammation of the knee. In normal fluids, no associations with age were observed. Antigen concentrations (uPA, tPA, and uPAR) in NGOA differed from those in other subclasses of OA, but the species of PA present did not appear to vary between disease groups. The predominant PA appeared to have identity with uPA. CONCLUSION: Because of the greater concentrations of these antigens in OA compared with normal fluids, OA cannot be used as a surrogate normal control in studies of the PA/PAI system. Alteration of the PA/PAI system was confirmed in RA and OA knee fluids, with greater changes evident in RA. The finding of different concentrations of PA antigens in NGOA compared with other OA fluids further supports a different pathogenic mechanism in this subset.     

Distribution of protein nitrotyrosine in synovial tissues of patients with rheumatoid arthritis and osteoarthritis

OBJECTIVE: Because nitric oxide related species have been found in the inflamed joints of patients with arthritis, we investigated whether protein nitrotyrosine (a marker of tissue exposure to peroxynitrite) is present in their synovial tissues. METHODS: Protein nitrotyrosine was detected immunohistochemically and by Western blot analysis. Synovial tissues removed surgically from 12 patients with rheumatoid arthritis (RA) (mean age 63.7 yrs) and 20 with osteoarthritis (OA) (mean age 66.6 yrs) were studied. RESULTS: Nitrated proteins were detected immunohistochemically in all of 18 tissues examined. Diffuse staining of the stroma was seen in all patients, with more extensive staining in RA than OA (p = 0.008). Intense staining was detected in some lymphocytes, but not in others, even within a single lymphoid aggregate. Neutrophils did not stain for nitrotyrosine. Vascular endothelial cells stained for nitrotyrosine but adjoining smooth muscle cells did not. Both cytoplasmic and nuclear staining was seen in macrophages, endothelial cells, and lymphocytes. Numerous bands of nitrated proteins were detected by Western blot analysis of 15 synovial tissue extracts. Inducible nitric oxide synthase (iNOS) was detected immunohistochemically in endothelial cells, macrophages, vascular smooth muscle cells, and synoviocytes. CONCLUSION: Nitrotyrosine-containing proteins were found in essentially all synovia from RA and OA patients. The most prominent site of nitration in all cases was the stroma. iNOS, the likely source of the nitrating species, was found in a variety of cell types.     

Plasminogen activator in synovial fluid from patients with rheumatoid arthritis

The plasminogen activator in synovial fluid from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) was analyzed on a molecular basis. The level of plasminogen activator in RA was found to be higher than in OA. The plaminogen activators of both RA and OA revealed 3 different molecular weights: 90,000, 55,000 and 33,000. RA demonstrated the 3 plasminogen activators in broadly comparable ratios, but OA had the 55,000 form dominantly. The 90,000 plasminogen activator was a tissue-type plasminogen activator, while the 55,000 and 33,000 plasminogen activators were of the urokinase-type. beta-Methasone suppressed the tissue-type plasminogen activator, and urinary trypsin inhibitor suppressed the urokinase-type plasminogen activators. When urinary trypsin inhibitor was injected clinically into the joint space of a patient with RA, the urokinase-type plasminogen inhibitor was suppressed as in the in vitro study, and the clinical signs and symptoms were markedly improved. Open trials of intraarticular injections of urinary trypsin inhibitor demonstrated improvement of the clinical signs and symptoms.     

Osteoprotegerin expression in synovial tissue from patients with rheumatoid arthritis,spondyloarthropathies and osteoarthritis and normal controls

OBJECTIVES: To demonstrate the expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappaB ligand (RANKL) in synovial tissue from rheumatoid arthritis (RA) patients, establish the cell lineage expressing OPG and compare the expression of OPG in RA, spondyloarthropathies, osteoarthritis and normal synovial tissue. METHODS: Synovial biopsy specimens were obtained at arthroscopy from 16 RA and 12 spondyloarthropathy patients with active synovitis of a knee joint, six RA patients with no evidence of active synovitis, 10 patients with osteoarthritis and 18 normal subjects. Immunohistological analysis was performed using monoclonal antibodies (mAb) to detect OPG and RANKL expression. In addition, dual immunohistochemical evaluation was performed with lineage-specific monoclonal antibodies (macrophages, fibroblasts and endothelial cells) and OPG to determine the cell lineages expressing OPG. The sections were evaluated by computer-assisted image analysis and semiquantitative analysis. RESULTS: Two patterns of OPG expression were seen, one exclusively in endothelial cells and one expressed predominantly in macrophages in the synovial lining layer. Both patterns of OPG staining could be blocked with excess recombinant OPG. Endothelial and synovial lining expression of OPG was seen in all synovial tissues except those from patients with active RA. In contrast, RANKL expression was seen predominantly in synovial tissue from patients with active disease, mainly in sublining regions, particularly within areas of lymphocyte infiltration. CONCLUSIONS: OPG expression on macrophage type synovial lining cells as well as endothelial cells is deficient in RA patients with active synovitis, in contrast to that seen in spondyloarthropathy patients with active synovitis. This deficiency in OPG expression in the inflamed joint of RA patients may be important in the development of radiologically defined joint erosions.     

Hepatocyte growth factor (HGF), HGF activator, and c-Met in synovial tissues in rheumatoid arthritis and osteoarthritis

OBJECTIVE: Hepatocyte growth factor (HGF) is a multifunctional polypeptide that has been implicated in cancer growth, tissue development, and wound repair. Its actions are dependent on activation by HGF activator (HGFA) and its binding to a specific HGF receptor (c-Met). We examined the role of HGF, HGFA, and c-Met in synovial tissues in rheumatoid arthritis (RA) and osteoarthritis (OA), and their localization and mRNA expression. METHODS: Immunohistochemical staining, Western blotting, RT-PCR, and in situ hybridization (ISH) for HGF, HGFA, and c-Met were performed on synovial tissue specimens from 10 patients with RA and 4 with OA, and 2 healthy controls. RESULTS: Immunohistochemical staining revealed that HGFA and c-Met were strongly expressed in fibroblasts, macrophages, endothelial cells, and synovial lining cells. HGF was expressed only faintly in macrophages and fibroblasts, and not at all in the endothelial cells of RA and OA synovial tissue. HGFA was detected near 73 and 34 kDa on Western blot analysis, corresponding to inactive and active HGFA, respectively. RT-PCR showed HGF, HGFA, and c-Met mRNA in RA, OA, and control synovial tissue. ISH and immunohistochemistry revealed mRNA expression for HGF, HGFA, and c-Met in the cell types mentioned above. CONCLUSION: HGFA, HGF, and c-Met mRNA are expressed in synovial tissue in RA and OA, and HGF is activated by HGFA and binds to c-Met on endothelial cells, inducing angiogenesis.     

Phospholipase activity in synovial fluid from patients with rheumatoid arthritis, osteoarthritis and crystal-associated arthritis

Phospholipase activity was assayed in cell-free synovial fluid (SF) from patients with rheumatoid arthritis (RA, n = 28), osteoarthritis (OA, n = 10), and crystal-associated arthritis (C, n = 7) by measuring the release of either [14C]oleic acid or [3H]arachidonic acid from radiolabeled E. coli phospholipids. Activity measured by oleic acid release was not significantly different between the three groups of patients (RA = 571 +/- 43.3, OA = 460 +/- 54.7 and C = 718 +/- 162.6 pmol/min/mg). Arachidonic acid release was significantly (p less than 0.005) less in OA (31 +/- 7.3) than RA (61 +/- 4.7) which was similar to C (58 +/- 17.6 pmol/min/mg). Arachidonic acid release correlated significantly with the SF white blood cell count (r = 0.483, p less than 0.01). This study shows the importance of the type of substrate used to measure phospholipase activity and indicates that differences in the capacity to release arachidonic acid may exist between RA and OA disease states.     

Release of cartilage and bone macromolecules into synovial fluid: differences between psoriatic arthritis and rheumatoid arthritis

OBJECTIVE: To elucidate whether differences in the destructive tissue process in cartilage and bone in psoriatic arthritis (PsA) and rheumatoid arthritis (RA) can be recognised by different release patterns of molecular fragments derived from joint tissue. METHODS: Aggrecan, cartilage oligomeric matrix protein (COMP), and bone sialoprotein (BSP) were quantified by immunoassays in knee joint synovial fluid samples. These were obtained early in the disease course of patients with PsA and RA. At the time of arthrocentesis radiographs of their knee and hip joints were normal. RESULTS: At follow up no destruction had developed in the knees and hips of most patients with PsA (n=18), whereas the patients with RA could be separated into one "destructive" group (n=18) and one "non-destructive" group (n=25). Patients with PsA had low synovial fluid aggrecan concentrations (p<0.001 v the RA destructive group) but high COMP concentrations (p<0.01 and p<0.05 v destructive and non-destructive RA groups, respectively). Consequently, the aggrecan/COMP ratio was lowest in the PsA group (p<0.001 and p<0.01 v the destructive and non-destructive RA group, respectively). The synovial fluid concentrations of BSP did not differ between the three patient groups. CONCLUSIONS: The release pattern of aggrecan and COMP, reflecting cartilage turnover, differed between the PsA group and, particularly, the destructive RA group. This suggests that different pathophysiological mechanisms for cartilage involvement operate in these conditions, with different destructive potential. The BSP concentrations did not differ between the patients groups, which indicates similar levels of bone involvement.     

Dickkopf-1 perpetuated synovial fibroblast activation and synovial angiogenesis in rheumatoid arthritis

Clinical Rheumatology - Dickkopf-1 (Dkk-1), a regulatory molecule of the Wnt pathway, is elevated and leads to bone resorption in patients with RA. This study is aimed to investigate the...     

Serum and synovial fluid histidine: a comparison in rheumatoid arthritis and osteoarthritis

Summary The serum and synovial fluid (SF) histidine, sulphydryl, and protein concentrations were compared in simultaneous samples from 84 patients with rheumatoid arthritis (RA) and a control group comprising 29 patients with osteoarthritis (OA). The SF levels of histidine were higher than the serum levels in the RA patients but significantly lower than corresponding results in patients with OA (P<0.001). The latter had levels of serum and SF histidine which were equivalent and within the normal range. Greater quantities of protein were found in the SF of the patients with RA compared with the OA group. The serum and SF sulphydryl concentrations expressed as mol/g protein were low but in equilibrium in patients with RA. However the SF sulphydryl (mol/g protein) was depressed relative to serum levels in patients with OA.     

Polyamine levels in synovial tissues and synovial fluids of patients with rheumatoid arthritis.

We determined the polyamine contents of the synovial tissues from 11 patients with rheumatoid arthritis (RA), and the free putrescine levels in the synovial fluids (SF) from 10 patients with RA, 7 with osteoarthritis (OA), 5 with posttraumatic arthritis, and 3 with infectious arthritis. Putrescine levels in the synovial tissues correlated with serum C reactive protein concentration in patients with RA. Free putrescine levels in SF were significantly elevated in patients with infectious arthritis, compared with those found in RA, OA, and posttraumatic arthritis. Free putrescine levels in SF from patients with RA were significantly higher than in those with OA. Our findings suggest that polyamines may play an important role in RA.     

Cysteine and serine proteases of synovial tissue in rheumatoid arthritis and osteoarthritis

OBJECTIVE: To compare the activities of cathepsin B (EC 3.4.22.1) and L (EC 3.4.22.15), calpain (EC 3.4.22.17), and dipeptidyl peptidase (EC 3.4.14.5 or DPP IV or CD26) in synovial membrane from patients with rheumatoid arthritis (RA), osteoarthritis (OA), and post-traumatic joint injury (PT). METHODS: Forty RA patients were divided into two groups on the basis of surgical procedure: the RAs group comprised 18 patients requiring surgical synovectomy; the RAr group comprised 22 patients requiring a total joint replacement or arthrodesis. A third group (the OA group) comprised 19 OA patients while six patients with post-traumatic joint injury were included in the fourth group (the PT group). Cathepsin and calpain activity was assessed using a Cobas Fara II centrifugal analyser. DPP IV activity was determined kinetically using a fluorogenic substrate. RESULTS: RAs patients were significantly younger than RAr patients, and the mean duration of RA was shorter in the RAs group than in the RAr group. Cathepsin and calpain activity in synovial membrane was higher in RA and OA patients than in the control group, but no statistical difference was observed between RA and OA. However, cathepsin, calpain, and DPP IV synovial activity was significantly higher in the RAs group than in either the OA or the PT group. CONCLUSION: Our results show that proteinase activity tends to be higher in joints with early synovitis in RA, and suggest that these enzymes are not all involved at the same stage of the disease.     

Early lymphocyte activation in the synovial microenvironment in patients with osteoarthritis: comparison with rheumatoid arthritis patients and healthy controls

Osteoarthritis (OA) is largely considered to be a non-inflammatory disease, although there is compelling evidence that subclinical inflammation is a common event, even in the absence of acute inflammatory flares. In this study we analyze, by means of CD5 and CD69 expression, the infiltration and early activation of CD5+cells, mostly lymphocytes, in both synovial membrane and synovial fluid from advanced OA patients and compare them with samples from patients with rheumatoid arthritis and healthy controls. The number of infiltrating CD5+ cells in both synovial membrane and synovial fluid from patients with advanced OA was significantly reduced as compared with rheumatoid arthritis patients. However, synovial membrane and synovial fluid CD5+ cells on OA exhibited a phenotype with evidence of recent activation comparable to that observed in RA.     

Analysis of cell populations in rheumatoid arthritis synovial tissues.

Knee synovium, taken from patients with rheumatoid arthritis at the time of arthroplasty, was studied immunohistologically. Focal perivascular lymphoid infiltrates of different sizes were examined in detail to evaluate changes in cell populations as the infiltrate size increased. T cells formed the largest component of mononuclear cells of all aggregates. The large grade 3 aggregates contained substantial numbers of B cells arranged around a central venule and cells bearing the CD45RA+ phenotype. In contrast, the small grade 1 aggregates contained few B cells and the T-cell population contained relatively greater numbers of CD8+ cells. Cells bearing the CD45RO+ phenotype exceeded CD45RA+ cells in grade 1 aggregates. Detailed analysis of mononuclear cell aggregates of different sizes in the rheumatoid synovium suggests that the composition of each aggregate depends on the total number of mononuclear cells it contains.     

Comparison of synovial tissues from the knee joints and the small joints of rheumatoid arthritis patients: Implications for pathogenesis and evaluation of treatment

OBJECTIVE: Serial synovial biopsy samples are increasingly being used for the evaluation of novel therapies for rheumatoid arthritis (RA). Most studies have used tissues from knee biopsies, but technical improvements have made serial small joint arthroscopy feasible as well. Theoretically, there could be differences in the features of synovial inflammation between various joints as a result of mechanical factors, differences in innervation, and other factors. We therefore undertook this study to compare the cell infiltrate in paired synovial biopsy samples from inflamed knee joints and paired inflamed small joints of patients with RA. METHODS: Nine RA patients with both an inflamed knee joint and an inflamed small joint (wrist or metacarpophalangeal joint) underwent an arthroscopic synovial biopsy of both joints on the same day. Multiple biopsy specimens were collected and stained for macrophages, T cells, plasma cells, fibroblast-like synoviocytes, and interleukin-6 (IL-6) by immunohistochemistry. Sections were evaluated by digital image analysis. RESULTS: There were no significant differences in mean cell numbers for all markers investigated in samples from the knee joint compared with samples from the small joints. We detected statistically significant correlations for the numbers of sublining macrophages, T cells, and plasma cells, as well as for IL-6 expression, between the knee joint and the small joints. However, there was no significant correlation between different joints for the numbers of intimal macrophages or fibroblast-like synoviocytes. CONCLUSION: The results of this study show that the inflammation in one inflamed joint is generally representative of that in other inflamed joints. Therefore, it is possible to use serial samples from the same joint, selecting either large or small joints, for the evaluation of antirheumatic therapies.     

Serum and synovial fluid adenosine deaminase activity in patients with rheumatoid arthritis, osteoarthritis, and reactive arthritis.

Adenosine deaminase activity was determined in paired samples of serum and synovial fluid taken from patients with rheumatoid arthritis (n = 12), reactive arthritis (n = 13), and osteoarthritis (n = 7), and the value of this investigation in the diagnosis of synovial swellings was assessed. Increased activity was found in the synovial fluid taken from patients with rheumatoid disease and reactive arthritis, though values were less raised in the latter. Synovial fluid taken from patients with osteoarthritis did not show significantly raised adenosine deaminase activity as compared with that of normal controls (n = 3).     

Comparison of blood and synovial fluid lymphocyte subsets in rheumatoid arthritis and osteoarthritis

Summary Immunoregulatory T-cell deficiency is thought to underlie pathogenesis of rheumatoid arthritis (RA) as a systemic autoimmunopathy. The aim of this study was a simultaneous analysis of peripheral blood and synovial lymphocyte subsets (Ly-SS) of RA patients as compared to patients with locally active osteoarthritis (OA). Peripheral blood Ly-SS and paired synovial fluid Ly-SS from 87 RA patients were analysed by two dimensional flow cytometry (Simulset Becton Dickinson) as compared to 15 OA patients. The control group consisted of 32 healthy subjects. The peripheral blood analysis from RA and OA patients revealed a significant decrease of CD8+T-cells and increase of CD4+:CD8+ ratio when compared to the control group. The blood of RA patients showed a significant increase of HLA DR+ and IL 2R+T cells as compared to OA group. The synovial fluid from RA and OA patients showed a significant increase of CD3+, CD8+, HLA DR+ T-cells and decrease of CD4+:CD8+ ratio and CD19+ cells in comparison to the peripheral blood. This study shows, that the OA T-cell system seems not to be activated in peripheral blood in opposition to RA patients. Synovial fluid Ly-SS in OA, however, showed only quantitative but not qualitative differences. OA seems to be mainly a local inflammatory response depending on T-cells, when lymphocyte T activity in blood is diminished.     

Bone resorbing activity in synovial fluids in destructive osteoarthritis and rheumatoid arthritis.

The synovial fluids of patients with a destructive form of osteoarthritis (DOA) were shown to contain high levels of bone resorbing activity as judged by the ability of the fluid to stimulate the release of 45Ca from labelled cultured mouse calvariae. The activity was lost on extended storage of the synovial fluids and was dependent for its effect on cellular activity in bone. Bone resorbing activity was present in most synovial fluids from patients with DOA and rheumatoid arthritis (RA) but occurred at higher levels in the former. In contrast, interleukin 1 (IL1) activity, measured by the mouse thymocytes costimulation assay, was higher in RA than DOA synovial fluids. Little or no bone resorbing or IL1 activity was detected in synovial fluids from patients with pseudogout or non-destructive osteoarthritis. These results suggest that most DOA synovial fluids contain a bone resorbing factor other than IL1. It is considered that the factor may be produced by synovial cells stimulated by hydroxyapatite crystals.