IL-6 in synovial fluids, plasma and supernatants from cultured cells of patients with rheumatoid arthritis and other inflammatory arthritides

The purpose of this investigation was to measure interleukin 6 (IL-6) levels in synovial fluid (SF) and plasma from patients with rheumatoid arthritis (RA) and other inflammatory arthritides (non-RA) and to examine the in vitro production of IL-6 by T cells and monocytes from SF and peripheral blood (PB). All patients had high levels of IL-6 in SF. Although the median level was higher in the group of non-RA arthritides, the difference was not statistically significant. Supernatants from both unstimulated and stimulated highly purified CD4+ and CD8+ cells from SF and PB did not contain IL-6, while high levels of IL-6 were detectable in supernatants of mononuclear cells (MNC) and plastic adherent cells without any specific in vitro stimulant. Since IL-6 is so readily produced by normal mononuclear cells in vitro without any specific stimulant, spontaneous in vitro production by SF MNC cannot be considered as evidence for in vivo production by the same cells.     

Correlation between synovial neopterin and inflammatory activity in rheumatoid arthritis.

According to recent investigations neopterin (a pyrazinopyrimidine derivative) is a biochemical marker that reflects the activity of the proinflammatory immunocellular system of the synovial tissue in rheumatoid arthritis (RA). Interferon gamma, derived from antigen activated T lymphocytes, stimulates macrophages to synthesise and release neopterin into the culture supernatant in vitro. To extend this in vitro model to a clinical level a sensitive new radioimmunoassay technique was used to measure neopterin concentrations in the synovial fluid (SF) of 17 patients with active RA, nine with osteoarthritis, and six with acute gout, and in that of 12 controls undergoing meniscectomy. The SF neopterin concentrations were significantly higher in patients with RA than in the other groups of patients, particularly the controls. Multivariant analysis showed that SF neopterin concentrations correspond better with the systemic inflammatory activity of RA than with the local disease activity of the knee joints. Thus the study strengthens the hypothesis that neopterin reflects the essential role of the activated immunocellular reaction in the pathogenesis of RA.     

Elevated C3 anaphylatoxin levels in synovial fluids from patients with rheumatoid arthritis

Because cleavage products of the third component of complement augment inflammation and may contribute to arthritis, we used a competitive inhibition radioimmunoassay to measure levels of the low molecular weight cleavage products of the third component of complement, C3a and C3adesArg, in 72 synovial fluid samples. Mean levels of C3a/C3adesArg were more than sevenfold higher in 41 patients who had rheumatoid arthritis than in 15 patients who had degenerative joint disease or 5 patients who had traumatic arthritis. Striking elevations were also present in 2 patients who had acute gouty arthritis. A calculation of the fraction of intraarticular C3 cleaved showed that the patients with rheumatoid arthritis had a mean C3 cleavage of 11.6 +/- 11.0%, which was significantly higher than values of less than 1.5% for patients with degenerative joint disease or traumatic arthritis. In rheumatoid arthritis and gouty arthritis, specific immunoassay identified substantial quantities of the initial C3 cleavage fragments.     

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.     

Platelet-activating activity in synovial fluids of patients with rheumatoid arthritis,juvenile rheumatoid arthritis,gout, and noninflammatory arthropathies

The predominant activating activity in rheumatoid synovial fluid for human platelets, as assessed by the release of ¹¹C serotonin, was previously isolated and shown to be a complex of IgG of molecular weight 450,000. Thus, a pooled 5 ml concentrate of the 450,000 molecular weight fraction obtained by Sepharose 66 gel filtration of 1 ml of synovial fluid was used to compare the platelet-activating activity (PAA) of fluids from 28 patients with inflammatory and noninflammatory ar-thropathies. One unit of PAA was arbitrarily defined as that volume of the 5 ml concentrate required to obtain release equal to that obtained with a standard dose of thrombin, and total units per milliliter of synovial fluid were calculated as the reciprocal of the volume of concentrate in milliliters containing one unit multiplied by five. A platelet-activating activity of >766 units/ml of synovial fluid was arbitrarily defined as elevated, since this value represents two standard deviations above the mean for the noninflammatory synovial fluids. Each of 7 noninflammatory fluids (range <50 to 695) and 3 of 4 gouty fluids (range <50 to 1,250) fell within the normal range, whereas 9 of 13 adult rheumatoid fluids (range <50 to 11,880), and 4 of 4 JRA fluids (range 1,299 to 5,100) had elevated levels of PAA per ml of synovial fluid. The finding that the purified synovial fluid 450,000 molecular weight complex of IgG was inhibited in its platelet-activating capacity by purified monoclonal rheumatoid factor suggests that a role for IgM rheumatoid factor could be to block IgG complex Fc receptor-dependent proinflammatory effects on platelets, and perhaps other cells.     

Glycosaminoglycans in the synovial fluids of patients with juvenile rheumatoid arthritis

A simple method was used to isolate hyaluronic acid (HA) and sulfated glycosaminoglycans from synovial fluids obtained from children with juvenile rheumatoid arthritis (JRA) and from normal age-matched controls. The bulk of the glycosaminoglycans present in both normal and pathologic synovial fluids consisted of hyaluronic acid and chondroitin 6-sulfate. The diseased synovial fluids showed a sharp decrease in the concentration of glycosaminoglycans when compared with normal controls. These findings are similar to those reported for the synovial fluid of adult patients with rheumatoid arthritis.     

Capacity to solubilize immune complexes in sera and synovial fluids from patients with rheumatoid arthritis

Fifty-six sera and 33 synovial fluids from rheumatoid arthritis patients were studied to evaluate their capacity to solubilize immune complexes. A decreased function was found in 65% of rheumatoid synovial fluids when compared with 13 osteoarthritis synovial fluids. The decrease of immune complex solubilization capacity correlated with the decrease of hemolytic activity of the alternative complement pathway (P < 0.001) and inversely with the level of immune complexes (P < 0.05). Synovial fluids with low and normal immune complex solubilization capacity were compared; the results confirmed a possible role of the synovial complement system in modulating the size and thus the diffusion and clearance of locally generated immune complexes.     

Correlation between synovial blood flow signals and serum vascular endothelial growth factor levels in patients with refractory rheumatoid arthritis

The objective of the study is to examine the relationship between synovial blood flow signals and vascular endothelial growth factor (VEGF) involved in angiogenesis by Doppler ultrasound. Twenty-one patients meeting the diagnostic criteria of the American College of Rheumatology (ACR) were enrolled in this study. Doppler ultrasound signals of blood flow in the wrist synovial membrane were measured and classified into three grades: grade 1 = no flow; grade 2 = mild flow; grade 3 = intense flow. A significant correlation was observed between blood flow signals in the wrist synovial membrane and serum VEGF levels (r = 0.5681, P = 0.0072). These results suggest that the measurement of Doppler ultrasound signals of blood flow in the wrist synovial membrane is useful in the evaluation of 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.     

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.     

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.     

Iron-binding proteins and free iron in synovial fluids of rheumatoid arthritis patients

Summary Iron-binding proteins (lactoferrin, transferrin and ferritin) and free iron were measured in synovial fluid (SF) from 30 patients with rheumatoid arthritis (RA) and 20 osteoarthritis (OA) patients. The iron-binding proteins except transferrin were significantly increased in RA SF as compared with OA SF. Similarly, free iron was also significantly higher in RA SF than in OA SF, whereas the ferritin saturation index, transferrin saturation index and bound iron were more significantly decreased in RA SF than in OA SF. These results suggest that RA SF contains sufficient micromolar amounts of free iron to allow hydroxyl radical formation. Also the capacity of iron-binding proteins to bind free iron is inadequate in the presence of a large amount of iron-binding proteins which are present in RA SF.     

Synovial fluids from patients with rheumatoid arthritis possessed B cell differentiation activity contributing to synthesis of rheumatoid factor in vitro

B cell differentiation activity assayed on B cells preactivated by Staphylococcus aureus Cowan I was found in synovial fluids (SF) of patients with rheumatoid arthritis (RA) (n = 14), while little, if any, activity was found in SF of patients without RA (n = 13) including patients with osteoarthritis and traumatic arthritis. There was a positive correlation between the RA hemagglutination assay titer and B cell differentiation activity (r = 0.7438). This molecule with molecular weight ranges 15-20 kDa as well as supernatant from phytohemaglutinin stimulated normal T cells could promote not only polyvalent immunoglobulin synthesis but also rheumatoid factor synthesis by lymphocytes from patients with RA in vitro, and was considered to be distinct from interleukin-1, interleukin-2, interferon r, or proteolytic lysosomal enzymes which have been shown to modulate B cell activation.     

Association between leptin and IL-6 concentrations with cardiovascular risk in patients with rheumatoid arthritis

Pro-inflammatory cytokines such as leptin and IL-6 play an important role in the development of cardiovascular risk. Determine the relationship between leptin and IL-6 concentrations with cardiovascular risk in patients with rheumatoid arthritis. We determined IL-6 and leptin levels in 77 patients with the diagnosis of rheumatoid arthritis. The cardiovascular risk was calculated using the modified Framingham scale. Statistical analysis was performed using SPSS 22 considering a significant p < 0.05. Serum leptin concentrations and cardiovascular risk (CVR) factors were compared and found that there was a significant difference between higher leptin values and disease activity (p 0.047), obesity (p 0.038), positive rheumatoid factor (p 0.009), tobacco (p 0.009), and metabolic syndrome (p 0.001). Likewise, a significant relationship was found between lower leptin concentrations and hydroxychloroquine consumption (p = 0.023). We found significant difference between IL-6 concentrations and disease activity (p 0.028), hypertriglyceridemia (p 0.023), LDL-C (p 0.029), and smoking (0.005). Similarly, an association between hydroxychloroquine consumption and low concentrations of IL-6 was found (p 0.005). Framingham CVR was calculated and the result obtained was multiplied by 1.5. The 35.2% of the population studied had a low Framingham CVR, 38.9% moderate, and 25.9% presented a high risk. We compared the level of CVR and serum leptin and IL-6 concentrations, finding that the highest CVR was the leptin and IL-6 values. There is a positive association between CVR and serum leptin concentrations. It is also significantly associated with traditional and non-traditional risk factors.     

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in synovial fluids from patients with rheumatoid arthritis or osteoarthritis

OBJECTIVE: Matrix metalloproteinases (MMPs) are expressed in joint tissues of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). The objective of this study was to define the steady state levels of seven different MMPs and two tissue inhibitors of metalloproteinases (TIMPs) as well as the potential metalloproteinase activity in the synovial fluid (SF) to provide more insight into the role of MMPs in cartilage destruction in RA and OA. METHODS: Levels of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-13, TIMP-1, and TIMP-2 in SF aspirated from knee joints of 97 patients with RA and 103 patients with OA were measured by the corresponding one step sandwich enzyme immunoassays. Proteolytic activity of MMPs in these SFs was examined in an assay using [(3)H]carboxymethylated transferrin substrate in the presence of inhibitors of serine and cysteine proteinases after activation with p-aminophenylmercuric acetate (APMA). Destruction of RA knee joints was radiographically evaluated. RESULTS: Levels of MMP-1, MMP-2, MMP-3, MMP-8, and MMP-9 were significantly higher in RA SF than in OA SF. MMP-7 and MMP-13 were detectable in more than 45% of RA SFs and in less than 20% of OA SFs, respectively. Among the MMPs examined, MMP-3 levels were extremely high compared with those of other MMPs. Direct correlations were seen between the levels of MMP-1 and MMP-3 and between those of MMP-8 and MMP-9 in RA SF. Although the levels of MMP-1 and MMP-3 increased even in the early stage of RA, those of MMP-8 and MMP-9 were low in the early stage and increased with the progression of RA. Molar ratios of the total amounts of the MMPs to those of the TIMPs were 5.2-fold higher in patients with RA than in OA, which was significant. APMA-activated metalloproteinase activity in SF showed a similar result, and a direct correlation was seen between the molar ratios and the activity in RA SF. CONCLUSIONS: Our results show that high levels of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, and TIMP-1 are present in RA SF and suggest that once these MMPs are fully activated, they have an imbalance against TIMPs, which may contribute to the cartilage destruction in RA.     

Sequential synovial fluid sampling suggests plasma and synovial fluid IL-6 vary independently in rheumatoid arthritis

SIR, Inflammatory cytokines, including interleukin-6 (IL-6),are raised in rheumatoid arthritis (RA). Single paired daytimesamples show that the IL-6 and TNF- concentrations in synovialfluid exceed blood concentrations during the day [1–3],and these and other data suggest that joints are the source.Recent data have shown that plasma IL-6 in RA follows a circadianpattern with a peak at 6 a.m. [4]. If IL-6 is generated insidejoints and diffuses into plasma, it might be expected that thesynovial fluid IL-6 concentration