The aminoterminal-type-III procollagen peptide and proteoglycans in serum and synovial fluid of patients with rheumatoid arthritis or reactive arthritis

Summary The concentrations of aminoterminal-type-III procollagen (procollagen N-) peptide, and of proteoglycans were measured in knee-joint synovial fluid and serum from patients with rheumatoid arthritis or reactive arthritis. All synovial fluids contained large amounts of intact propeptide. The synovial fluid : serum propeptide ratios were high, suggesting local propeptide liberation. A correlation was demonstrated between the propeptide concentration in synovial fluid and in serum. In rheumatoid arthritis, the propeptide concentration in synovial fluid was related to local inflammatory activity, and the serum concentration was correlated with the presence of nonspecific markers of inflammation. The presence of smaller propeptide fragments in synovial fluid indicated that some degradation occurred locally. The local metabolic changes were most prominent in patients with joint erosions. Patients with nonerosive rheumatoid arthritis and reactive arthritis had similar synovial fluid propeptide concentrations. The proteoglycan content of synovial fluid was inversely related to the degree of joint destruction, and was highest in patients with reactive arthritis. No correlation was observed between the concentrations of propeptide and proteoglycan in synovial fluid. Intra-articular glucocorticoid injection reduced the levels of propeptide and proteoglycan in synovial fluid.     

Induction of a lymphotoxin-like mediator in peripheral blood and synovial fluid lymphocytes by incubation with synovial fluid from patients with rheumatoid arthritis

Summary A significantly increased spontaneous cell-mediated cytotoxicity (SCMC) has been reported in synovial fluid lymphocytes (SFL) as compared to peripheral blood lymphocytes (PBL) of patients with rheumatoid arthritis (RA) and that of normal controls [1–3]. To determine whether this increased SCMC activity is due to the production of a lymphokine and related to the production of a lymphotoxin (LT)-like mediator, PBL from normal controls and PBL and SFL from RA patients were incubated either with a human melanoma cell line (IGR 3) or with cell-free synovial fluid (SF) from RA patients. The SF and the cell-free supernatants of the different cultures were tested for LT activity by estimation of inhibition of DNA synthesis of HeLa cell monolayers and they were added to a SCMC assay system using normal PBL and IGR 3 as target.In the supernatants from cocultures of either PBL from controls or PBL and SFL from RA patients with IGR 3 cells, there was no significant difference in LT activity. An LT-like mediator was observed in the supernatants of all lymphocytes cocultured with SF, whereas SF alone and supernatants of lymphocytes alone exhibited little or no LT activity. In a control experiment, LT induction was not observed when normal lymphocytes were cultured with the serum of RA patients. Absorption of the culture supernatants with an insolubilised goat anti-human Ig did not remove LT activity. The demonstrated release of an LT-like mediator from lymphocytes incubated with SF might be one contributing mechanism to the inflammatory joint reaction in RA patients.     

Identification of citrullinated cellular fibronectin in synovial fluid from patients with rheumatoid arthritis

Abstract

Objectives. Cellular fibronectin (cFn) has been implicated in the pathogenesis of rheumatoid arthritis (RA), and we previously demonstrated the presence of citrullinated cFn in rheumatoid synovial tissues. The present study aimed to investigate whether citrullinated cFn can be detected in the plasma or synovial fluid of RA patients.

Methods. Twenty-five rheumatoid arthritis synovial fluid (RASF), seven osteoarthritis synovial fluid (OASF) and 12 plasma samples from RA patients were examined. Citrullination of cFn was determined by immunoprecipitation (IP), western blotting and enzyme-linked immunosorbent assay (ELISA), in which peptidyl-citrulline within cFn was detected using a specific anti-cFn monoclonal antibody in combination with anti-modified citrulline antibody after chemical modification.

Results. Levels of citrullination associated with cFn, as determined by ELISA, were significantly higher in RASF than in OASF samples. IP and western blotting detected citrullinated cFn in RASF but not in plasma samples from RA patients. Levels of total cFn were elevated in RASF compared with OASF, and 24 out of 25 RASF samples were positive for anti-CCP antibody. However, no correlation was observed between levels of citrullinated cFn and those of total cFn or anti-CCP antibody in RASF. On the other hand, a significant positive correlation was observed between the levels of matrix metalloproteinase-3 (MMP-3) and cFn citrullination in RASF.

Conclusions. Citrullinated cFn appears to be produced within the affected joint and might be involved in the pathogenesis of rheumatoid synovitis.     

Plasma and synovial fluid concentrations of sulphasalazine and two of its metabolites in rheumatoid arthritis

Summary A study was made of plasma and synovial fluid levels of sulphasalazine, one of its dissociation products — sulphapyridine and a metabolite of the latter — acetyl sulphapyridine in patients with rheumatoid arthritis (RA) who were in a steady state on sulphasalazine therapy. Combined sulphapyridine levels were significantly higher than those of sulphasalazine both in plasma and synovial fluid. Synovial fluid levels of both drugs correlated with their plasma levels and were generally slightly lower. Some patients accumulated sulphasalazine and sulphapyridine in the synovial fluid and the mean concentration of sulphasalazine was higher in the fluid than in the plasma. The explanation for this is uncertain. The concentration of combined sulphapyridine in synovial fluid was related to local joint inflammation and more active systemic disease. No consistent association was found between sulphasalazine levels and local or systemic activity. The higher sulphapyridine levels in synovial fluid found in this study suggest the possibility that this moiety could play a more active role in RA than it does in inflammatory bowel disease.     

Interleukin-6 in synovial fluid is closely associated with chronic synovitis in rheumatoid arthritis

Summary Interleukin-6 (IL-6) was detected at low levels in plasma [0.014±0.006 ng/ml (mean ± SEM] and in high amounts in synovial fluid [SF; 2.6±2.2 ng/ml (mean ± SEM)] of patients with rheumatoid arthritis. No correlation of IL-6 levels in plasma or SF with the ESR (n=15) or with histological parameters of acute local synovitis (n=10) was observed. In contrast, SF IL-6 was positively correlated with histological characteristics of chronic synovitis (n=10; P0.01) and elevated plasma IgG concentrations (n=15; P0.05). In vitro concentrations of IL-6 comparable to those detected in SF increased the production of both IgG and IgM by synovial membrane mononuclear cells. The present results contribute to the view that high local IL-6 concentrations in SF promote chronic synovitis in RA.     

Serum and synovial fluid leptin levels and markers of inflammation in rheumatoid arthritis

This study was designed to investigate the serum and synovial fluid leptin levels, and inflammatory markers in rheumatoid arthritis (RA) patients. Serum and synovial fluid leptin levels were significantly higher (P > 0.05) in RA patients than control group; RA patients with moderate disease activity (DAS < 2.7) having significantly higher leptin levels (P > 0.05) than those with low disease activity (DAS < 2.7). Leukocytes and erythrocyte sedimentation rate (ESR) were found to be significantly higher in moderate disease activity RA group compared to low activity group (P > 0.05, P < 0.001, respectively). Serum leptin level is found to be independent of age and inflammatory markers. ESR is positively correlated with DAS activity and CRP values. Our finding of no correlation between leptin and BMI shows that regulation of leptinemia is complex, and leptin levels cannot be used to assess RA activity.     

Evidence for activation of platelets in the synovial fluid from patients with rheumatoid arthritis

Summary Platelets were isolated by gel filtration from paired samples of peripheral blood and synovial fluid (SF) aspirated from inflamed knee joints from 20 adult patients with rheumatoid arthritis (RA) as well as from peripheral blood obtained from 20 healthy subjects. The platelets from the three different sources were investigated for quantitative differences in the number of two distinct types of intracellular storage organelles using immunofluorescence staining for platelet factor 4 (PF4) and labelling with the fluorescent substance mepacrine (MC). The number of PF4-stained organelles per cell was the same in the peripheral normal and RA platelets. This number was distinctly lower in the SF platelets. The peripheral and SF platelets from the RA patients had the same number of MC-labelled organelles. This number was distinctly lower than in the normal cells. The results suggest that the peripheral RA platelets had been activated to liberate serotonin and other substances from one type of organelles, and that the SF platelets had been activated to an additional liberation of PF4 from another such type. Liberated PF4, serotonin, and other substances from SF platelets may, in several ways, contribute to the inflammatory responses of RA.     

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.     

Comparison of modern marker proteins in serum and synovial fluid in patients with advanced osteoarthrosis and rheumatoid arthritis

Numerous studies have focused on the significance of modern marker proteins in the synovial fluid of the knee joint and in the serum both, for osteoarthritis (OA) and rheumatoid arthritis (RA). The relationship between the serum concentrations and the concentrations in the synovial fluid is still unclear. Synovial fluid and serum samples were obtained from 13 patients with advanced OA and from 8 patients with severe RA and concentrations of MMP-1, MMP-3, MMP-13, TIMP-1, COMP and MIA/CD-RAP were determined. All values were normalized against the total protein concentrations. Serum concentrations of MMP-13 in the RA-group were statistically higher than the synovial values (P<0.05). MMP-13 was the only marker protein that revealed distinct higher levels in the serum than in the synovial fluid. The study design allows only conclusions about advanced stages of RA and OA. Longitudinal investigations may provide further information about the value of MMP-13 as a potential marker to monitor the course of RA and OA.     

Cathepsin G and elastase in synovial fluid and peripheral blood in reactive and rheumatoid arthritis

Summary The purpose of the study was to evaluate the involvement of serine proteinases cathepsin G and elastase on pathomechanisms in synovial fluid (SF) of patients with reactive (ReA) and rheumatoid, (RA) arthritis. Cathepsin G, elastase, and their endogenous inhibitors ₁-antichymotrypsin (₁-ACT) and ₁-proteinase inhibitor (₁-PI) were identified immunohistochemically from SF and peripheral blood (PB) of patients with ReA and RA. Cathepsin G and elastase activities in SF and PB were measured spectrophotometrically. Dot-immunostaining was used to identify cathepsin G, elastase, but also ₁-ACT and ₁-PI from SF and PB. Cathepsin G and elastase-like activities (IU/I) were slightly elevated in ReA SF compared to the corresponding peripheral blood values (11.4±9.2 vs 4.8±1.7, NS, and 5.1±2.8 vs 2.3±2.2, NS), which was similar to what was seen in RA (16.4±6.2 vs 0.53±0.4, p<0.05, and 6.51±1.8 vs 1.22±0.58, p<0.05). Although some samples did not contain cathepsin G and/or elastase-like activities, all samples contained immunoreactive enzyme, but also ₁-ACT and ₁-PI. In ReA SF, in contrast to monocytes, all polymorphonuclear (PMN) cells contained cathepsin G and elastase. Cathepsin G and elastase activities correlated with each other (r=0.78, p<0.05) suggesting PMN / primary granules as their likely source. There was a closer association between the cathepsin G or elastase and SF leukocyte count in ReA than in RA. In ReA and RA SF elevated cathepsin G and elastase activities are detected compared to activity levels in PB suggesting local production mainly from PMNs. The co-existence of highly cellular SF and cathepsin G and elastase activity in the documented presence of endogenous inhibitors in ReA SF together with the, known, usually self-remitting clinical course of ReA, suggest a brisk and even exaggerated local PMN serine proteinase release; sparing of joints does not seem to be due to lack or inhibition of PMN responses but rather to a successful down-regulation or cessation of the responses initially elicited.     

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.     

Kynurenic acid, an endogenous constituent of rheumatoid arthritis synovial fluid, inhibits proliferation of synoviocytes in vitro

Kynurenic acid is an antagonist of ionotropic glutamate receptors. It has been found that glutamate antagonists inhibit proliferation of different human tumor cells. Since the hyperplasia of synovial fibroblasts is one of the most striking features of inflammatory arthritis, the main goals of this study were detection and quantification of kynurenic acid in synovial fluid obtained from patients with rheumatoid arthritis, and determination of its effect on proliferation of synoviocytes in vitro. Presence of kynurenic acid was determined by HPLC in all 58 samples of synovial fluid. The mean concentration was 15.89 pmol/ml. Kynurenic acid inhibited synoviocyte proliferation with the IC₅₀ value of 5.9 mM. In subthreshold concentration of 0.3 mM it enhanced antiproliferative action of celecoxib and nimesulide. In conclusion, the presence of kynurenic acid in synovial fluid was documented in patients with rheumatoid arthritis. Its potential role as an endogenous substance, controlling synoviocyte proliferation can be suggested.     

Interleukin 1 beta in synovial fluid is related to local disease activity in rheumatoid arthritis

Summary Interleukin 1 beta (IL-1 beta) is a polypeptide with pro-inflammatory and immunopotentiating effects in vivo and in vitro. With relevance to rheumatoid arthritis (RA) IL-1 augments release of prostanoids, proteinases and oxygen metabolites and is a potent inducer of bone and cartilage resorption. Although high levels of IL-1 have been found in rheumatoid synovial fluids, intra-individual variation in IL-1 production has made it difficult to correlate these levels with disease activity. To overcome this problem we have studied patients with symmetrical and asymmetrical knee joint inflammation. Local disease activity was documented using Ritchie score and joint circumference; IL-1 beta levels were quantitated in synovial fluid by ELISA. In patients with symmetrical joint involvement almost identical levels of IL-1 beta were detected in the right and left knee joints. In contrast, in patients exhibiting asymmetrical knee joint involvement, IL-1 beta levels in the inflamed joints were significantly higher than in the contralateral joints. The study provides further evidence for the role of IL-1 in the pathogenesis of rheumatoid inflammation.     

Leukocyte count in the synovial fluid of children with culture-proven brucellar arthritis

Brucellosis is an important cause of paediatric septic arthritis in endemic areas. Because the Gram stain is frequently negative and culture results are unavailable at the time of the patient’s admission, the diagnosis of brucellar arthritis is usually entertained on the bases of epidemiological considerations and cytological examination of the synovial fluid aspirate. The aim of this study was to assess the sensitivity of a synovial fluid leukocyte count >50 000 WBC/mm³ for detecting culture-proven brucellar arthritis in children. The medical records of all children with brucellar arthritis diagnosed since 1994 in a hospital serving an endemic area for brucellosis in southern Israel were reviewed. Nine patients (six males and three females), aged 3–14 years, were identified. A single joint was affected in all patients. The median leukocyte count in the synovial fluid was 9500 WBC/mm³ (range 300–61 500 WBC/mm³), and in eight of the nine patients it was less than 50 000 WBC/mm³. Brucella melitensis was recovered from the synovial fluid culture in all patients. The diagnosis of brucellar septic arthritis cannot be excluded on the basis of a low leukocyte count in the joint aspirate. A high index of suspicion and use of modern culture techniques are recommended to improve the diagnosis of brucellar arthritis. Received: 24 March 2001 / Accepted: 29 October 2001     

Quantitative analysis of pyridinium crosslinks of collagen in the synovial fluid of patients with rheumatoid arthritis using high-performance liquid chromatography

The pyridinium crosslinks are important and definite biomarkers of mature hard tissue collagen degradation. A gradient ion-paired reversed-phase high-performance liquid chromatographic method was used for the simultaneous determination of both crosslinks in synovial fluid (SF) samples of 25 patients with rheumatoid arthritis (RA). The mean±SD levels of pyridinoline (Pyd) and deoxypyridinoline (Dpyd) in SF were 107.7±182.3 nmol/1 and 4.8±8.3 nmol/1, respectively. The Pyd/Dpyd ratio, which indicates the amount of Pyd released from cartilage rather than bone, amounted to 30.8±29.5. This value is significantly higher than in urine or serum of the same patients. These data suggest increased destruction of joint cartilage in patients with RA and the release of collagen II fragments in SF. In addition, the levels of the crosslinks in SF reflect considerable interindividual variation, indicating substantial individual differences in the amount of collagenous material that is degraded.     

T cell subsets in the blast cell population in phytohemagglutinin-stimulated peripheral blood in vitro and in rheumatoid arthritis synovial fluid in vivo

Summary The immunomodulatory T4/T8 ratio was studied in the total and activated lymphocyte populations by a method combining visualisation of ³H-thymidine incorporating blasts with autoradiography (AR) with simultaneous identification of the respective lymphocyte subsets using monoclonal antibodies in avidin-biotin-peroxidase complex (ABC) staining. In rheumatoid arthritis synovial fluid the activated T4/T8 ratio (calculated from T cells in the S phase of the cell cycle) was significantly different from the total T4/T8 ratio (calculated for all the T cells) (0.45±0.05 versus 0.69±0.05, P<0.01). Similarly, the activated and total T4/T8 ratios were also significantly different in the phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cell cultures at days 3 and 5.     

Two-dimensional electrophoresis of synovial tissue,synovial fluid and serum in patients with rheumatoid arthritis and related diseases

Summary Using the technique of two-dimensional (2D) electrophoresis with consecutive silver staining, we investigated samples of serum, synovial fluid and synovial tissue obtained from 19 patients suffering from rheumatoid arthritis (RA) or non-RA arthritis. From these experiments we have drawn the following conclusions. 2D electrophoresis of serum, synovial fluid and synovial tissue extracts taken from patients suffering from joint diseases is a reproducible method. Repeated runs of the same sample reveal an essentially constant protein spot pattern. The time period between surgery and tissue preparation did not influence the number of protein spots when less than 15 h was involved. The protein spot number is always lower in synovial fluid than in either synovial tissue or serum in RA and non-RA patients. The mean value for the number of spots is 68 for the inflamed tissue irrespective of the cause of arthritis (RA and non-RA group taken together) and 47 for the control group. This difference is significant. We were able to definitely identify 7 spots in the tissue extract. We did not find RA-specific protein spots in either serum, synovial fluid or tissue extracts from the synovial membrane. The only significant difference between RA patients and either non-RA or control group patients concerning the protein spot pattern is the increased size of the immunoglobulin spot (mainly IgG) in RA. In addition, we discuss possible reasons for failure of the 2D electrophoresis technique to detect disease-specific protein patterns.     

Infiltrations of plasma cells in synovium are highly associated with synovial fluid levels of APRIL in inflamed peripheral joints of rheumatoid arthritis

Infiltration of plasma cells can be a histopathological hallmark of articular synovium with rheumatoid arthritis (RA). A proliferation-inducing ligand (APRIL) may have key roles in homeostasis and development of B cells, and the differentiation of B cells into plasma cells. This study was designed to explore the relationships between the infiltrations of plasma cells in synovium and the synovial fluid levels of APRIL in inflamed peripheral joints of RA. Synovium and synovial fluid were sampled from 21 RA patients underwent arthroscopic synovectomy for inflamed peripheral joints. The variants of rheumatoid synovium were classified into the follicular and diffuse synovitis by hematoxylin and eosin staining, and the infiltrations of plasma cells in rheumatoid synovium were quantified under the light microscope. The synovial fluid levels of APRIL were measured with the enzyme-linked immunosorbent assay. The mean number of infiltrating plasma cells in synovium and the mean synovial fluid level of APRIL were significantly increased in follicular synovitis compared with those in diffuse synovitis (P = 0.009, and P = 0.018, respectively), and there was a highly positive association between the infiltrations of plasma cells and the synovial fluid levels of APRIL among all of the RA patients (Rs = 0.776, P < 0.001). These findings suggest that the local production of APRIL may be associated with the ectopic lymphoid neogenesis in rheumatoid synovium and may have a role in contributing to the infiltration of plasma cells in synovium within inflamed peripheral joints of RA.     

Infections and arthritis

Bacteria, viruses, fungi, and parasites can all cause arthritis of either acute or chronic nature, which can be divided into infective/septic, reactive, or inflammatory. Considerable advances have occurred in diagnostic techniques in the recent decades resulting in better treatment outcomes in patients with infective arthritis. Detection of emerging arthritogenic viruses has changed the epidemiology of infection-related arthritis. The role of viruses in the pathogenesis of chronic inflammatory arthritides such as rheumatoid arthritis is increasingly being recognized. We discuss the various causative agents of infective arthritis and emphasize on the approach to each type of arthritis, highlighting the diagnostic tests, along with their statistical accuracy. Various investigations including newer methods such as nucleic acid amplification using polymerase chain reaction are discussed along with the pitfalls in interpreting the tests.     

Analysis of childhood reactive arthritis and comparison with juvenile idiopathic arthritis

There is currently no agreement on how to classify and diagnose reactive arthritis (ReA) and what kind of clinical and laboratory findings are specific for the diagnosis. This study retrospectively analyzed the initial clinical manifestations and laboratory findings in children diagnosed with ReA and juvenile idiopathic arthritis (JIA). A comparison was also made between these two groups to see if there were differences. A retrospective chart review was performed and 44 patients diagnosed with ReA and 80 patients with JIA were enrolled in this study. Their initial clinical manifestations and laboratory findings were also analyzed and compared. The initial clinical manifestations in ReA were analyzed including the demographic data, the preceding infection history, the duration of the infectious episode to the onset of arthritis, the duration of arthritic symptoms, and the involved joint pattern. Comparison of the initial laboratory findings between patients with ReA and JIA showed significant differences between erythrocyte sedimentation rates (ESR) in the first hour, platelet counts (p<0.05), and ESR in the second hour (p=0.052). Further, comparing ReA with the subtypes of JIA, significant differences were noted between ReA and the systemic type in terms of hemoglobin level, platelet counts, C-reactive protein, and first and second hour ESR (p<0.05). However, if compared with the polyarticular or pauciarticular type, only the platelet counts showed any significant statistical difference (p<0.05). This study summarizes clinical experiences in ReA. The differences in laboratory findings of ReA and JIA may provide a clue in making a differential diagnosis.