Complexes In Synovial Fluid Research Articles

Identification of a complex between fibronectin and aggrecan G3 domain in synovial fluid of patients with painful meniscal pathology

Objectives We previously described a panel of four cytokines biomarkers in knee synovial fluid for acute knee pain associated with meniscal pathology. The cytokine biomarkers included interferon gamma (IFN-γ), interleukin 6 (IL-6), monocyte chemotactic protein 1 (MCP-1), and macrophage inflammatory protein-1 beta (MIP-1β). Validation studies using other immunologic techniques confirmed the presence of IL-6, MCP-1 and MIP-1β, but not IFN-γ. Therefore we sought the identity of the IFN-γ signal in synovial fluid. Methods Knee synovial fluid was collected from patients with an acute, painful meniscal injury, as well as asymptomatic volunteers. A combination of high-pressure chromatography, mass spectrometry and immunological techniques were used to enrich and identify the protein components representing the IFN-γ signal. Results A protein complex of fibronectin and the aggrecan G3 domain was identified in the synovial fluid of patients with a meniscal tear and pain that was absent in asymptomatic controls. This protein complex correlated to the IFN-γ signal. A novel enzyme-linked immunosorbent assay (ELISA) was developed to specifically identify the complex in synovial fluid. Conclusions We have identified a protein complex of fibronectin and aggrecan G3 domain that is a candidate biomarker for pain associated with meniscal injury.

Soluble oligonucleosomal complexes in synovial fluid from inflamed joints.

To determine whether soluble oligonucleosomal DNA, typical of that released during apoptotic cell death, is present in synovial fluids from inflamed joints and, if so, whether it is present in sufficient concentrations to have pathophysiologic significance. Fifty synovial fluid specimens from 46 patients were studied, 41 from joints with a variety of inflammatory disorders and 9 from osteoarthritic joints. DNA from freshly collected synovial fluid was isolated and quantitated by microfluorometry, and the oligonucleosomal fraction was measured by radiolabeling, gel electrophoresis, and autoradiography. Specific immunoprecipitation with monoclonal antihistone antibody, after DNA radiolabeling in whole synovial fluid, was used to detect histone binding. DNA with a typical oligonucleosomal ladder was observed in most specimens. The mean +/- SD oligonucleosomal DNA concentration was 14.1 +/- 18.5 microg/ml in synovial fluids from inflamed joints, considerably higher than that in osteoarthritic synovial fluids. Additionally, the DNA was shown to be complexed with histone, as would be expected. Control experiments were performed to show that the oligonucleosomal DNA was present in soluble form and did not arise due to in vitro artifact. The DNA concentrations were found to correlate significantly with the concentrations of synovial fluid leukocytes, most of which were neutrophils. Synovial fluids from inflamed joints contain oligonucleosomal DNA typical of that released during apoptotic cell death. The probable source is fluid-phase neutrophils undergoing apoptotic cell death, although this was not directly demonstrated. The concentrations are sufficient to have biologic activity similar to that shown in vitro, including lymphoproliferation and stimulation of interleukin-6 secretion. A mechanism by which oligonucleosomal DNA may contribute to perpetuation of rheumatoid synovitis is proposed. If it is generalizable to other sites of inflammation, as seems probable, similar oligonucleosomal DNA release accompanying inflammation may play a pathogenetic role in other disorders, including systemic lupus erythematosus.

Fibrin degradation in the synovial fluid of rheumatoid arthritis patients: a model for extravascular fibrinolysis.

The patterns of degradation and the influence of factor XIII polymerization on fibrin stability were examined in vitro following incubation with leukocyte elastase. In vivo experiments, various factor XIII-polymerized fibrin clots were implanted subcutaneously in mice to evaluate the stability of clots in the extravascular space. Both in vitro and in vivo lysis proceeded faster with nonpolymerized fibrin and was not influenced by the presence of cross-linked alpha 2-plasmin inhibitor. In vivo lysis of implanted clots was prevented by elastatinal, powerful elastase inhibitor, suggesting that granulocyte elastase is chiefly responsible for clot lysis in the extravascular space. To further extend investigations on the mechanisms of fibrinolysis in tissues, we evaluated fibrin and its degradation products in the synovial space. Expression of factor XIII in synovial cells and activities of coagulation factors, fibrinolytic enzymes, and inhibitors were investigated in the synovial fluid of rheumatoid arthritis patients. Immunohistochemical analysis showed deposits of insoluble fibrin on synovial membranes and pannus to an extent related to the progression of the disease. Factor XIII was expressed by fibroblasts and macrophages in the early stages of the disease, whereas in advanced stages factor XIII staining was associated with fibrin. The reduction of certain coagulation factors and high level of thrombin-antithrombin complexes in synovial fluid show a steady activation of the coagulation cascade. The evaluation of fibrinogen degradation products and the pattern of degradation of synovial fibrin(ogen) suggest the participation of leukocyte elastase in fibrin(ogen) lysis in synovial tissue of rheumatoid arthritis.

Intraarticular α2-Macroglobulin Complexes and Proteolytic Activity in Children with Juvenile Rheumatoid Arthritis

In juvenile rheumatoid arthritis (JRA), it is likely that the release of proteolytic enzymes from activated synovial fluid neutrophils overwhelms the major protease inhibitor, alpha 2-macroglobulin (alpha 2-MG), and leads to cartilage destruction. Due to the unique nature of the alpha 2-MG-protease complex, proteolytic function is maintained until the complex is cleared. In this study, we sought to determine the concentration of alpha 2-MG-protease complexes in synovial fluid of patients with JRA, the proteolytic activity found in their synovial fluid, and whether the alpha 2-MG complexes are associated with increased proteolytic activity. The JRA patients' synovial fluids had complex levels of 217.0 +/- 192.2 nmol/L--significantly elevated compared with plasma values (p < 0.001) and with control synovial fluid (p < 0.05). Elastase activity (almost entirely neutrophil elastase) was detected in all JRA synovial fluid samples (mean 2.9 +/- 2.6 mg/L) and significantly correlated with alpha 2-MG-complex values (r = 0.67, p < 0.01). Synovial fluid tryptic activity was detectable in all JRA patients but did not significantly correlate with alpha 2-MG complexes (r = 0.53, p > 0.05). Seventy-four percent of total elastase activity and 41% of total tryptic activity were contained in the alpha 2-MG-complex fractions. We suggest that the increased concentration of synovial fluid alpha 2-MG complexes with retained elastase activity contributes to continued proteolysis and joint destruction and may affect the subsequent disease course through its role as a modulator of IL-6.

The measurement of collagenase, tissue inhibitor of metalloproteinases (TIMP), and collagenase-TIMP complex in synovial fluids from patients with osteoarthritis and rheumatoid arthritis.

To compare the levels of collagenase, tissue inhibitor of metalloproteinases (TIMP), and collagenase-TIMP complex in synovial fluid (SF) from patients with rheumatoid arthritis (RA) and patients with osteoarthritis (OA). This study aims to clarify existing data from previously used enzyme or inhibitor activity assays performed following separation by gel filtration, by using a 1-step enzyme-linked immunosorbent assay (ELISA) for each component. Total collagenase, free TIMP, and collagenase-TIMP complex were measured using a newly developed, specific double-antibody sandwich ELISA: Levels of both collagenase and TIMP were significantly higher in RA patients (collagenase 1,560 +/- 150 ng/ml [mean +/- SEM], TIMP 1,610 +/- 130 ng/ml; n = 80) than OA patients (collagenase 420 +/- 90 ng/ml, TIMP 1,050 +/- 60 ng/ml; n = 80), with the difference being especially striking for collagenase. Sixteen RA fluids had detectable levels of collagenase-TIMP complex, compared with only 3 OA fluids. The level of total collagenase in SF is greater in RA than OA, while levels of free TIMP show more overlap between the 2 diseases; this may simply reflect the increased inflammation seen in RA, or it may reflect a different disease mechanism.

Analysis of intraarticular fibrinolytic pathways in patients with inflammatory and noninflammatory joint diseases.

Intraarticular activation of the fibrinolytic system has been suspected to occur in patients with arthritis. We undertook the present study to investigate the relation of this activation to clinical symptoms, and the molecular pathways involved. We quantitatively assessed levels of plasmin-alpha 2-antiplasmin (PAP) complexes in synovial fluid (SF) from 25 patients with rheumatoid arthritis (RA), 7 with seronegative spondylarthropathy (SSA), and 10 with osteoarthritis (OA), and conducted an analysis to determine the plasminogen-activating pathway via which these complexes were generated. In addition, we studied the relationship of intraarticular fibrinolysis to clinical and biochemical parameters. All patients studied had increased SF levels of PAP complexes. Levels in patients with RA and SSA were slightly higher than those in patients with OA. These complexes were probably formed by activation of urokinase-type plasminogen activator (u-PA), and not tissue-type plasminogen activator (t-PA), since SF levels of both u-PA antigen and u-PA-plasminogen activator inhibitor (PAI) complexes were increased in 27 of the 42 patients. Conversely, SF levels of t-PA were below normal in all but 1 patient. In some patients, activation of factor XII presumably also contributed to plasminogen activation in SF, since levels of factor XIIa-C1 inhibitor in SF were increased in 8 of the 42 patients and correlated, as did u-PA-PAI levels, with levels of PAP complexes. Several of the parameters of fibrinolysis in SF, particularly u-PA antigen and u-PA-PAI-1 complexes, were found to correlate with clinical and biochemical parameters. Our results suggest that plasminogen is frequently activated in the joints of patients with inflammatory or noninflammatory arthropathy and that this activation mainly occurs via a u-PA-, and in some cases also via a factor XII-, dependent pathway. The possible relation of this activation process to stimulation of synovial cells by cytokines is discussed.

IgE-containing immune complexes in synovial fluid of patients with rheumatoid arthritis

The prevalence and composition of IgE-containing immune complexes in paired synovial fluid and serum of 42 patients with classical or definite rheumatoid arthritis were studied. IgE-containing immune complexes were found in 15/42 synovial fluids; 15 sera were also positive. The correlation between serum and synovial fluid complexed IgE levels was high (r = 0.77). The mean ratio of synovial fluid/serum levels was 1.96, i.e. significantly higher than 0.33, the synovial fluid/serum ratio for alpha-2-macroglobulin (molecular weight 820 kD), which was taken as high molecular weight control protein (p less than 0.0001). Apart from IgE in immune complex form, monomeric IgE was also significantly higher in synovial fluid compared to serum (ratio = 2.94). Other constituents which could be found in the immune complexes, i.e. anti-IgE antibodies, rheumatoid factors and anticollagen antibodies, were also higher in synovial fluid than predicted. Our results suggest intra-articular production of IgE-containing complexes in the synovial fluid, in addition to possible exudation of the complexes from the serum. These findings provide further evidence for the role of IgE-containing immune complexes in rheumatoid synovitis.

Rheumatoid factors in immune complexes of patients with rheumatoid arthritis.

Substantial evidence indicates that humoral immunity through antigen-antibody complex formation contributes to the pathogenesis of RA. Several studies have examined the composition of immune complexes present in the synovial fluid of patients with RA. The constituents of these immune complexes have been immunoglobulins and complement components. Only a few polypeptides in these immune complexes have not been identified. These unidentified components account for only a few percent of total proteins present. These results suggest that rheumatoid factors and their antigens are quantitatively the important ingredients of immune complexes in synovial fluid of patients with RA. The immune deposits in the superficial layers of articular cartilage from patients with RA contain RFs and antibodies to type II collagen.

Teichoic acid antibody measurement as an aid to the diagnosis of septic arthritis due to Staphylococcus aureus

Hitherto laboratory diagnosis of septic arthritis has been dependent on Gram films, bacteriological culture and leucocyte counts with varying amounts of success. Patients with septic arthritis, rheumatoid arthritis and other conditions in which joint inflammation is significant have been analysed for the presence of specific staphylococcal teichoic acid antibody (TAA) in serum and synovial fluid specimens as an alternative to provide improved diagnosis. Good correlation between a positive bacterial culture for Staphylococcus aureus and TAA was seen in those patients with staphylococcal septic arthritis. Serum proved to be a better source of the antibody than synovial fluid. Thepresence of immune complexes in synovial fluid may account for this difference in sensitivity.

Immune complexes in synovial fluid and serum from patients with disseminated gonococcal infection: evidence for local immune complex formation within the joint.

Twenty one patients with acute arthritis associated with disseminated gonococcal infection (DGI) were studied. Synovial fluid (SF) from 14 and serum from 15 (matched in eight) were assayed for the presence of immune complexes (IC) by the Raji cell immunofluorescent assay (Raji IFA) and the 125I-Clq polyethylene glycol (PEG) binding assay. Higher levels and frequency of IC were detected in the SF by both IC assays and these were associated with a significant increase in complexes containing IgM over serum (p less than 0.02). Complexes containing IgG were found predominantly in serum and were infrequent in SF (p less than 0.003). These data suggest that the arthritis of DGI may result from primary immune complex formation within the synovial cavity after local antibody synthesis within the synovium in response to gonococcal seeding.

IgG and IgM antibody to native type II collagen in rheumatoid arthritis serum and synovial fluid. Evidence for the presence of collagen-anticollagen immune complexes in synovial fluid.

Levels of IgG and IgM antibody to native type II collagen, a component of articular cartilage, were measured by a specific solid-phase radioimmunoassay, in 48 rheumatoid arthritis patients. Good correlations were found between the levels in sera and those in synovial fluids. Collagenase digestion caused a significant rise in specific antibody levels in synovial fluids, indicating the presence of intraarticular collagen-anticollagen complexes. These immune complexes may be one mechanism for perpetuation of inflammation in some rheumatoid arthritis patients.

Immune Complexes in Rheumatoid Arthritis Sera and Synovial Fluids

Sera and synovial fluids from 88 patients with rheumatoid arthritis were examined for circulating immune complexes by three assays: monoclonal rheumatoid factor radioimmunoassay, C1q binding assay, and Raji cell radioassay. Paired samples were available for 82 patients. Immune complexes were detected with high frequency in the synovial fluid by each assay (75% by the monoclonal rheumatoid factor radioimmunoassay, 95% by the C1q binding assay, and 61% by the Raji cell readioassay). In rheumatoid arthritis sera, immune complexes were detected with high frequency by the C1q binding assay (85%) and the monoclonal rheumatoid factor radioimmunoassay (70%) but infrequently by the Raji cell radioassay (26%). The presence of immune complexes in serum was most frequently accompanied by the presence of complexes in fluid, regardless of the method of detection; moreover, the levels of immune complexes in synovial fluid were generally higher than in paired serum. Further, the levels of immune complexes as measured by the C1q binding assay correlated with certain parameters of clinical activity, while the monoclonal rheumatoid factor radioimmunoassay and Raji cell radioassay correlated with extraarticular features (excluding nodules) of rheumatoid arthritis.

The primary plasmin inhibitor in rheumatoid synovial fluid.

In 20 consecutive rheumatoid arthritis patients, 14 women and 6 men, age 26--76, average 62 years, the concentration of the recently found "primary plasmin inhibitor" and phase proteins was estimated in plasma and synovial fluid. In 12 patients a complex between the inhibitor and plasmin could be demonstrated by crossed immunoelectrophoresis into immunoglobulins against the primary plasmin inhibitor and immunoglobulin against plasminogen. Only free inhibitor was found in corresponding plasma. All plasminogen present in synovial fluid could be activated to plasmin upon addition of urokinase (24 nM/1). In those patients where enzyme-inhibitor complex in synovial fluid was present, a higher concentration of phase proteins in synovial fluid was found, indicating an increased degree of inflammation despite identical scores in the Lansbury clinical index in the two groups. From these experiments it was concluded that the fibrinolytic capacity in rheumatoid synovial fluid is not decreased. It is suggested that the fibrin-like material in synovial tissue and upon the synovial membrane is a poor substrate for plasmin.

Immunologically induced acute synovitis in rabbits. Studies of immune complexes in synovial fluid.

AbstractLarge antigen‐antibody complexes that are selectively removed from circulation were not selectively removed when injected into the knees of a normal rabbit. The subsequent selective removal of the complexes depended on the influx of neutrophils. Nevertheless, macrophages of the normal synovial lining were able to phagocytize some immune complexes. When antigen and antibodies were administered separately by intravenous or intraarticular routes, little or no soluble complexes were found in synovial fluid, and most of the antigen‐antibody union was thought to occur in the synovial tissue.

The dimensions of the particle of hyaluronic acid complex in synovial fluid.

The dimensions of the particle of hyaluronic acid complex in synovial fluid.