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Synovial fluid analysis plays an important role in the differential diagnosis of various forms of arthritis. The results of macroscopic, biochemical, bacteriological, and microscopic techniques of evaluating synovial fluid must be correlated to establish the diagnosis of each of the various forms of arthritis. The principle of polarized microscopy as applied to the differential diagnosis of crystalline arthritis is currently of special interest.
OBJECTIVE: To examine mononuclear cell (MNC) subtypes in osteoarthritis (OA) synovial fluid (SF) and compare these results with MNC subtypes in SF from patients with rheumatoid arthritis (RA). METHODS: MNC were obtained from SF by gradient centrifugation with Ficoll-Hypaque. MNC subtypes were identified using color immunofluorescence with monoclonal antibody pairs and flow cytometry. RESULTS: Significantly lower percentages of CD3+ (p < 0.01) and CD4+ cells (p < 0.01) were found in patients with OA. No differences were noted between CD8+ cells and the CD4/CD8 ratio. Significantly higher percentages of CD14+ (p < 0.001) and CD16+/CD56+ (p < 0.001) were found in patients with OA. CONCLUSION: Significant differences in MNC subtypes in OA and RA were noted that may reflect differences in pathophysiologic mechanisms operational in each disease.
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OBJECTIVE: To evaluate in vitro migration of mononuclear cells towards synovial fluid (SF) and plasma in relation to RANTES synovial fluid levels and clinical disease activity. METHODS: 31 RA patients with synovitis in one knee were included. Modified Boyden chamber technique was used to determine a migratory index defined as: 'In vitro migrating cells towards SF' divided by 'In vitro migrating cells towards plasma'. RANTES was quantified by ELISA. Disease activity was assessed by the swollen joint count, the Ritchie articular index (RAI), global assessment, pain on VAS, HAQ, ESR and CRP. RESULTS: A positive significant correlation was found between the migratory index and the RANTES levels in SF (r=0.48, p=0.006), the RAI (r=0.56, p=0.0001) and pain on VAS (r=0.43, p=0.04). The in vitro migration could be inhibited in 3 of 4 SF samples by neutralising antibodies towards RANTES (12-18%). CONCLUSION: The migratory index correlate to SF levels of RANTES and parameters for joint pain.
Normal synovial membranes and synovial membranes from patients with classic rheumatoid arthritis were investigated for the presence of fibrin and fibronectin by an indirect immunoperoxidase technique. In normal synovial membranes, fibronectin was found around the monolayer of the synovial lining cells. Staining was most intense on the surface and beneath the lining cells, but not detectable in the cytoplasm. Fibronectin was also found in the cytoplasm of the endothelial cells. No staining for fibrin was found in the normal synovial membrane. In synovial membranes from patients with rheumatoid arthritis, large amounts of fibronectin were found around the multilayer of synovial lining cells, in the cytoplasm of the endothelial cells, and in argyrophilic fiber-rich connective tissue. In superficial areas denuded of synovial lining cells, high amounts of fibronectin were found incorporated in fibrin. In some areas with noninjured synovial lining cells, fibrin was also found, but in this case no fibronectin was incorporated. No fibronectin was found in connective tissue in areas with infiltration of inflammatory cells. After treatment of normal and rheumatoid synovial membranes with hyaluronidase, fibronectin was still present around the lining cells but the staining was found to be more distinct. This study relates the presence of fibrin and fibronectin in the rheumatoid synovial membrane to the high amount of these proteins, recently described, in rheumatoid synovial fluid. It also suggests that fibronectin present in the synovial membrane is produced and secreted by the endothelial cells.
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In rheumatoid arthritis (RA) a chronic inflammatory state exists in which the synovial fluid is periodically filled with large numbers of polymorphonuclear leukocytes (PMNs). Oxygen radicals produced by these cells have been implicated as mediators of tissue damage and may be directly involved in the pathogenesis of RA. We examined the production of oxygen radicals by synovial fluid PMNs (SF-PMNs) and peripheral blood PMNs (PB-PMNs) by measuring chemiluminescence (CL) as well as superoxide anion (O2-) release. Increased spontaneous CL in the presence of luminol and increased CL in response to phorbol myristate acetate (PMA) was observed in SF-PMNs when compared to PB-PMNs. When zymosan was used as the stimulus in the absence of luminol, a slightly lower CL response was observed in SF-PMNs as compared to PB-PMNs. No significant differences were observed in the generation of O2- generation with any stimulus. Preincubation of normal PB-PMNs in 10% synovial fluid enhanced the luminol-dependent spontaneous and PMA-stimulated CL as well as zymosan-stimulated CL. When O2- release from normal PB-PMNs pretreated with 10% synovial fluid was compared to untreated controls, enhancement of spontaneous O2- release was observed. PMA- and zymosan-stimulated responses did not differ significantly from controls. Increased spontaneous and PMA-stimulated release of myeloperoxidase (MPO) was also observed in normal PB-PMNs pretreated with synovial fluid. These findings may explain the increased luminol-dependent CL since this type of CL requires the presence of MPO. Our findings suggest that the enhanced chemiluminescence observed in normal PMNs treated with synovial fluids may be related to increases in spontaneous O2- generation and myeloperoxidase release. Increased MPO release may account for enhanced CL observed in SF-PMNs.
OBJECTIVE: To assess the influence of inflammatory synovial fluid (SF) on apoptosis of joint and blood neutrophils with particular reference to levels of colony stimulating factors (CSF) contained therein. METHODS: Neutrophils were separated from fresh synovial fluid and from peripheral blood by density gradient centrifugation. Apoptosis was assayed by light microscope morphology and DNA degradation. CSFs were assayed using bone marrow bioassay and enzyme linked immunosorbent assays for granulocyte (G-) and granulocyte macrophage (GM-) CSF. Separated neutrophils were cultured in vitro and exposed to: varying concentrations of SF in which CSF levels were measured, recombinant G-CSF and GM-CSF, and hyaluronic acid control solutions. Numbers of apoptotic neutrophils and CSF levels were also measured in fresh SF samples. RESULTS: The addition of autologous or heterologous inflammatory SF to blood or joint cavity neutrophils cultured in vitro caused a significant dose dependent increase in the percentage of cells becoming apoptotic with time as measured morphologically and confirmed by DNA degradation. The effect bore no relationship to levels of CSF in joint fluid, despite our finding that GM-CSF produced inhibition of neutrophil apoptosis in vitro. CONCLUSION: These data suggest that SF contains a factor or factors capable of directly or indirectly promoting neutrophil apoptosis and normally powerful enough to overcome the apoptosis inhibiting effects of cytokines such as GM-CSF at concentrations usually found in inflammatory synovial fluids.
The effect of synovial fluid and washings of synovial membrane on autologous lymphocytes from patients with rheumatoid arthritis and other diseases has been studied using a rapid method based upon the increase in intranuclear birefringence occurring in the early stages of lymphocyte activation. Retardation of polarized light indicating increased lymphocyte activation was seen in lymphocytes from patients with rheumatoid arthritis but not in lymphocytes from patients with other diseases.
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.
Synovial fluid (SF) accumulates in the joint cavity in different conditions; this review outlines the data from those analyses that help in their differential and definitive diagnosis. The gross appearance of the fluid can provide a quick bedside orientation with regard to the amount of inflammation present in the joint: totally transparent SF originates in non-inflammatory conditions--of which osteoarthritis is the most common--and the amount of turbidity grossly relates to the amount of inflammation. Most turbid to purulent fluids usually come from infected joints, but exceptions are not uncommon. The white cell count offers quantitative information, but the boundaries between non-inflammatory and inflammatory SF and between this and septic fluid are very hazy and figures have to be interpreted in the clinical setting. Detection and identification of monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystals allow a precise diagnosis of gout and CPPD crystal-related arthropathy. Only one in five CPPD crystals have sufficient birefringence for easy detection and they are easily missed if searched for only using a polarised microscope. Instructions for beginners are given. Proper microbiological studies of the SF is the key to the diagnosis of infectious conditions.
The synovial cavity constitutes the ideal stage to study the interplay between microbial Toll-like receptor (TLR) ligands and cytokines. Infiltrated leukocytes and synovial fibroblasts produce cytokine- and chemokine-induced proteases for remodeling the extracellular matrix. The regulation of chemokine function for attraction and activation of leukocytes constitutes a key feature in host immunity and resolution of inflammation after infection. Enhanced levels of the CXC chemokine ligand (CXCL9)/monokine induced by interferon-gamma (IFN-gamma) and CXCL11/IFN-inducible T cell alpha chemoattractant, two chemoattractants for activated T cells and natural killer cells, and ligands for CXC chemokine receptor 3 (CXCR3) were detected in the synovial fluid of septic arthritis compared with osteo- and crystal arthritis patients. In vitro, IFN-gamma and TLR3 ligation by double-stranded RNA (dsRNA) induced the expression of CXCL9 and CXCL11 in leukocytes and skin-muscle fibroblasts, whereas ligation of TLR2, TLR4, TLR5, and TLR9 by peptidoglycan (PGN), lipopolysaccharide (LPS), flagellin, and unmethylated CpG oligonucleotides, respectively, did not. PGN and LPS, but not unmethylated CpG oligonucleotides, even inhibited IFN-gamma-induced CXCL9 and CXCL11 expression in leukocytes. In sharp contrast, in fibroblasts, the TLR ligands PGN, dsRNA, LPS, and flagellin synergized with IFN-gamma for the production of CXCL9 and CXCL11. Although TLR ligands stimulate leukocytes to produce CXCL8/interleukin-8 during the early innate defense, they contribute less to the production of CXCR3 ligands, whereas fibroblasts are important sources of CXCR3 ligands. These results illustrate the complex interaction between cytokines and TLR ligands in infection.
Rheumatoid synovial fluid contains an activator of latent collagenase from culture medium of pig synovium. The activator was purified by gel chromatography on Ultrogel AcA 44 and affinity chromatography on soybean trypsin inhibitor coupled to Sepharose 4B. The purified material was homogeneous on SDS-polyacrylamide gel electrophoresis with Mr 88 000. The activator had limited proteolytic activity against azo-casein, but showed amidase activity on Pro-Phe-Arg-NMec, Z-Phe-Arg-NMec, D-Val-Leu-Arg-NPhNO2 and D-Pro-Phe-Arg-NPhNO2, with an optimum at pH 8.0. Activity was completely inhibited by diisopropyl fluorophosphate, soybean trypsin inhibitor, leupeptin and Pro-Phe-Arg-CH2Cl, whereas lima bean trypsin inhibitor, Tos-Lys-CH2Cl, a specific inhibitor of factor XIIa from maize, EDTA and iodoacetate were not inhibitory. These properties of the activator suggested that it might be plasma kallikrein (EC 3.4.21.34), and the possibility was further examined. The activator was treated with [3H]diisopropyl fluorophosphate, and run in SDS-polyacrylamide gel electrophoresis with reduction; a radioautograph of the gel showed a pair of [3H]diisopropyl phosphoryl-labelled bands (Mr 36 000 and 34 000) identical to those obtained with authentic plasma kallikrein. Double immunodiffusion with monospecific antiserum against human plasma kallikrein confirmed the identification. This is the first demonstration of collagenase-activating activity of plasma kallikrein, and raises the possibility that activation of prokallikrein in the inflamed joint space may contribute to the disease process not only by the production of bradykinin, but also by activating latent collagenase.
OBJECTIVE: To measure synovial fluid (SF) levels of interleukin-1 receptor antagonist (IL-1ra) and to determine the capacity of SF neutrophils (PMN) to synthesize and release IL-1ra. METHODS: A sensitive and specific enzyme-linked immunosorbent assay was used to measure SF IL-1ra protein concentrations and IL-1ra production by isolated SF PMN: RESULTS: SF IL-1ra levels were elevated in 13 of 16 samples from patients with rheumatoid arthritis (RA) (mean 17.1 ng/ml), in 6 of 18 samples from patients with infectious or inflammatory, non-RA arthropathies (mean 10.6 ng/ml), and in none of 11 noninflammatory SF samples. SF IL-1ra levels correlated with SF PMN concentrations (r = 0.680, P < 0.00001). Isolated SF PMN contained preexisting IL-1ra protein in the absence of messenger RNA (mRNA). In addition, both lipopolysaccharide and granulocyte-macrophage colony-stimulating factor induced modest increases in IL-1ra mRNA by cultured SF-PMN. CONCLUSION: IL-1ra levels are increased in > 80% of RA SF samples. SF PMN produce IL-1ra, possibly contributing to the levels of IL-1ra present within the SF.
Synovial fluid was aspirated from the knees of 125 patients and lipid profiles were determined. The patients had knee injuries with or without fracture of bone; these lipid profiles were compared with "controls" (synovial fluid obtained at surgery from patients that did not have a knee injury). Floating lipid droplets were observed in some of the synovial fluid from patients with fractures. These lipid droplets could be separated as a well-defined supernatant layer after a few minutes of centrifugation (100 X g). This layer was found to consist mostly of triglycerides. The synovial fluid from patients with fracture and those with only soft-tissue trauma showed increases in total lipids and triglyceride content but had lower phospholipids when compared with nontraumatized knees. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin, cholesterol, monoglycerides, diglycerides, triglycerides, and free fatty acids were present in all synovial fluids assayed. The large amount of triglycerides in the synovial fluid in many cases may be a good indicator of marrow leakage. Because the soft tissue surrounding the knee is also rich in triglycerides, a low phospholipid level in some cases may be an even better indicator of marrow leakage.
OBJECTIVE: To determine the levels of hepatocyte growth factor (HGF) in synovial fluids (SF) and sera from patients with rheumatoid arthritis (RA); to examine how these correlate with several disease variables in patients with RA and with levels of interleukin-6 (IL-6) in SF of these patients; and to examine whether HGF is released from adherent synovial cells (ASC) and synovial fluid cells (SF cells). METHODS: An enzyme linked immunosorbent assay was used to measure levels of HGF and IL-6. SF samples were obtained from 22 patients with RA, 12 with osteoarthritis (OA), and one with septic arthritis. Serum samples were collected from 40 patients with RA. HGF levels in culture supernatants from ASC and SF cells were measured. RESULTS: The mean values of HGF in SF were 1.21 ng/ml for patients with RA, 0.19 ng/ml for those with OA and 0.18 ng/ml for the one with septic arthritis. HGF levels in SF of patients with RA were significantly higher than of those with OA (p < 0.01). The levels for patients with RA correlated with the serum C-reactive protein concentrations (r = 0.626, p < 0.01) and IL-6 levels in SF (r = 0.476, p < 0.05). The mean value of HGF in sera from patients with RA was 0.28 ng/ml. HGF levels in SF were higher than those in sera drawn simultaneously from the same patients with RA. In vitro, release of HGF from rheumatoid ASC was not detected. However, SF cells from patients with RA released HGF spontaneously. CONCLUSION: Our observations suggest that HGF in SF of patients with RA is produced by SF cells and is related to disease activity of RA, and thus that HGF may play a role in RA.
The immunopathology of inflamed joints in patients with RA is thought to result from an antigen-driven T-cell response. The antigen(s) responsible for the activation of synovial T cells, however, are as yet unidentified. In this study, we tested SF as a potential source of (auto)antigen(s). Five of 15 IL-2-expanded T-cell lines generated from SF cells of RA patients displayed a proliferative response to autologous SF. Five CD4+CD8-alpha beta TCR+SF-reactive T-cell clones obtained from responder T-cell lines were studied in more detail. Three T-cell clones from one RA patient were found to recognize epitopes in autologous SF in the context of DR4(Dw4), and two T-cell clones of another RA patient responded to autologous SF in the context of the HLA-DPB1*0401 gene product. The two DP-restricted clones and one of the DR-restricted clones did not proliferate to 50 SF samples of other RA patients, whereas the remaining DR-restricted clones responded to one allogeneic sample. Sequence analysis demonstrated that the latter clones expressed identical V beta 6.9 + TCR beta chains. This was also found for the (V beta 19+) DP-restricted clones. Proliferation of SF-reactive T cells was not only obtained with SF of the joint that had contained the T cells, but also with autologous SF of other affected joints. Together, these findings indicate that epitopes able to stimulate synovial T cells differ among RA patients, but may be similar within multiple joints of an individual patient. The presence of T cells able to respond to SF antigens in inflamed joints suggests that these T cells play an active role in the pathogenesis of RA.
The non-synovial tendon was placed in the synovial cavity of rabbit knee joint to carry out tissue culture in vivo. The surface of the cultured tendon was covered with a smooth membrane-like tissue which possessed the morphologic characteristics of the synovial membrane identified by histological and electron microscopic examinations. The tendon segments were kept free in the cavity of knee joint and adhesions were not observed. It is suggested that non-synovial tendon could transform into a synovial tendon in the environment filled with synovial fluid.