The metabolism leukotriene B4 in synovial fluid and whole human blood

The metabolism of synthetic leukotriene B₄ (LTB₄) in synovial fluid from rheumatoid arthritis and osteoarthritis patients and in whole blood from these same patient groups and from normal volunteers has been studied. A linear relationship existed between a plot of the time of incubation of samples with LTB₄ and the percentage of the initial concentration of LTB₄ at each time point. The slope of this line, the rate constant for metabolism, has been used to compare different samples. LTB₄ was metabolised more rapidly in the synovial fluid of rheumatoid arthritis patients than osteoarthritis patients. Furthermore, LTB₄ was metabolised more rapidly in the blood of rheumatoid arthritis patients that either osteoarthritis patients or normal volunteers. These differences in metabolism correlate with the polymorphonuclear leukocyte (PMN) and albumin content of samples. It is suggested that binding of LTB₄ to albumin will in part determine the available concentration of LTB₄ in inflammatory lesions.     

Leukotriene B4 and prostaglandin E2-like activity in synovial fluid in osteoarthritis

LTB4 and PGE2-like activity in synovial fluid samples from patients with osteoarthritis of the knee joint were determined and found to be significantly higher than in samples obtained from normal patients. The results suggest that leukotrienes and prostaglandins may have a role in the pathogenesis of osteoarthritis.     

Carboxypeptidase N (kininase I) activity in blood and synovial fluid from patients with arthritis

Carboxypeptidase N (CPN, kininase I) and kininase II (angiotensin converting enzyme) activities were measured simultaneously in blood plasma and synovial fluid in patients suffering from rheumatoid arthritis (RA), psoriatic arthritis (PA) and osteoarthritis (OA) and in the plasma of normal volunteers. CPN levels (defined as the rate of hydrolysis of furylacryloyl-Ala-Lys) in blood were modestly increased and correlated with erythrocyte sedimentation rate in RA and PA. Based on the hydrolysis of synthetic substrates, CPN activity was much higher than kininase II activity in synovial fluid (SF). SF kininase activities were always inferior to the blood levels in all patients and were correlated with the logarithm of SF leukocyte counts, an indicator of the intensity of inflammation. In addition, CPN and albumin levels in SF were highly correlated when expressed as a percent of the plasma concentrations. Biochemical properties of CPN in crude SF confirmed its similarity to blood CPN. Polymorphonuclear leukocytes derived from inflammatory SF did not release CPN. It is concluded that kininases diffuse from the blood into SF through increased vascular permeability and that CPN could be a major metabolic pathway for kinins in this form of exudate. CPN leads to the formation of des-Arg kinins, selective agonists of the B1 receptors for kinins.     

Chemokine receptor CCR4 on CD4+ T cells in juvenile rheumatoid arthritis synovial fluid defines a subset of cells with increased IL-4:IFN-gamma mRNA ratios

To understand the mechanisms that promote recruitment and survival of T cells within the pediatric inflamed joint, we have studied the expression of CCR4 and CCR5 on synovial fluid T cells and matched peripheral blood samples from juvenile rheumatoid arthritis (JRA) patients using three-color flow cytometric analysis. Thymus- and activation-regulated chemokine and macrophage-derived chemokine, ligands for CCR4, were measured by ELISA in JRA synovial fluid, JRA plasma, adult rheumatoid arthritis synovial fluid, and normal plasma. IL-4 and IFN-gamma mRNA production was assessed in CD4+/CCR4+ and CD4+/CCR4(-) cell subsets. We found accumulations of both CCR4+ and CCR5+ T cells in JRA synovial fluids and a correlation for increased numbers of CCR4+ T cells in samples collected early in the disease process. Thymus- and activation-regulated chemokine was detected in JRA synovial fluid and plasma samples, but not in adult rheumatoid arthritis synovial fluid or control plasma. Macrophage-derived chemokine was present in all samples. CD4+/CCR4+ synovial lymphocytes produced more IL-4 and less IFN-gamma than CD4+/CCR4(-) cells. These findings suggest that CCR4+ T cells in the JRA joint may function early in disease in an anti-inflammatory capacity through the production of type 2 cytokines and may play a role in determining disease phenotype.     

Application of a novel protein biochip technology for detection and identification of rheumatoid arthritis biomarkers in synovial fluid

We compared protein profiles of the synovial fluid of patients with rheumatoid arthritis and osteoarthritis by using surface-enhanced laser desorption/ionization mass spectrometry technology. With this approach, we identified a protein expressed specifically in the synovial fluid of the patients with rheumatoid arthritis. During the investigation, we found several reproducible and discriminatory biomarker candidates for distinction between rheumatoid arthritis and osteoarthritis. Among these candidates, a 10 850 Da protein peak was the clearest example of a single signal found specifically in the rheumatoid arthritis samples. This candidate was purified using a size-exclusion spin column followed by gel electrophoresis and subsequently identified by peptide mapping and post-source decay (PSD) analysis. The results clearly indicate that the protein is myeloid-related protein 8, which was verified by the enzyme immunoassay. It is known that the myeloid-related protein 8 level in serum and synovial fluid is related to disease activity in juvenile rheumatoid arthritis. The results suggest that the ProteinChip platform is useful to detect and identify protein biomarkers expressed specifically in diseases or in some stage of diseases.     

High levels of IL-17 in rheumatoid arthritis patients: IL-15 triggers in vitro IL-17 production via cyclosporin A-sensitive mechanism

Recent data suggest that IL-15 plays an important role in the pathogenesis of rheumatoid arthritis. In the present study, we hypothesized that elevated in the joints of rheumatoid arthritis, but not osteoarthritis, patients, IL-15 may exert its proinflammatory properties via the induction of IL-17, a cytokine known to stimulate synoviocytes to release several mediators of inflammation including IL-6, IL-8, GM-CSF and PGE2. To test this hypothesis, we first measured the levels of IL-17 and IL-15 using specific ELISA and found that synovial fluids of patients with rheumatoid arthritis, but not with osteoarthritis, contain high levels of these cytokines. A strong correlation between IL-15 and IL-17 levels in synovial fluids was observed. Among tested factors, LPS and TNF-alpha failed, IL-15 and IL-2 were equipotent, and PMA + ionomycin was far more efficient in the induction of IL-17 secretion by PBMCs isolated from healthy blood donors. Interestingly, synovial fluid cells, in contrast to PBMCs isolated from patients with rheumatoid arthritis, but not osteoarthritis, respond to PMA + ionomycin with much lower, comparable to IL-15-triggered IL-17 secretion. Moreover, PMA + ionomycin-triggered IL-17 secretion is completely or partially blocked in the presence of low doses of cyclosporin A or high doses of methylprednisolone, respectively. IL-15-triggered IL-17 secretion by PBMCs was completely inhibited by these drugs. Thus, our results suggest for the first time that IL-15 may represent a physiological trigger that via cyclosporin A and steroid sensitive pathways leads to the overproduction of IL-17 in the joints of rheumatoid arthritis patients.     

Enhanced levels of leukotriene B(4) in synovial fluid in Lyme disease

The purpose of this study was to evaluate the potential role of LTB(4) and cysteinyl leukotrienes in Lyme disease (LD). Therefore, a total number of 34 patients divided into four groups was studied. The patients were classified as having Lyme arthritis (n = 7) or Lyme meningitis (n = 10), and as control groups patients with a noninflammatory arthropathy (NIA) (n = 7) and healthy subjects (n = 10). LTB(4) as well as LTC(4) secretion from stimulated polymorphonuclear leukocytes (PMNL) from all groups of patients showed no statistical differences. LTB(4) levels in synovial fluid were significantly increased in patients with Lyme arthritis (median 142 ng/ml, range 88-296) when compared to the control subjects with NIA (median 46 ng/ml, range 28-72) (p < 0.05). No statistical difference of urinary LTE(4) levels between all the different groups of patients was observed. These results show that cysteinyl leukotrienes do not play an important role in the pathogenesis of LD. In contrast to previous findings in rheumatoid arthritis, LTB(4) production from stimulated PMNL was not found to be increased in LD. However, the significantly elevated levels of LTB(4) in synovial fluid of patients with Lyme arthritis underline the involvement of LTB(4) in the pathogenesis of this disease.     

Early rheumatoid arthritis is characterized by a distinct and transient synovial fluid cytokine profile of T cell and stromal cell origin

Pathological processes involved in the initiation of rheumatoid synovitis remain unclear. We undertook the present study to identify immune and stromal processes that are present soon after the clinical onset of rheumatoid arthritis (RA) by assessing a panel of T cell, macrophage, and stromal cell related cytokines and chemokines in the synovial fluid of patients with early synovitis. Synovial fluid was aspirated from inflamed joints of patients with inflammatory arthritis of duration 3 months or less, whose outcomes were subsequently determined by follow up. For comparison, synovial fluid was aspirated from patients with acute crystal arthritis, established RA and osteoarthritis. Rheumatoid factor activity was blocked in the synovial fluid samples, and a panel of 23 cytokines and chemokines measured using a multiplex based system. Patients with early inflammatory arthritis who subsequently developed RA had a distinct but transient synovial fluid cytokine profile. The levels of a range of T cell, macrophage and stromal cell related cytokines (e.g. IL-2, IL-4, IL-13, IL-17, IL-15, basic fibroblast growth factor and epidermal growth factor) were significantly elevated in these patients within 3 months after symptom onset, as compared with early arthritis patients who did not develop RA. In addition, this profile was no longer present in established RA. In contrast, patients with non-rheumatoid persistent synovitis exhibited elevated levels of interferon-γ at initiation. Early synovitis destined to develop into RA is thus characterized by a distinct and transient synovial fluid cytokine profile. The cytokines present in the early rheumatoid lesion suggest that this response is likely to influence the microenvironment required for persistent RA.     

Elevated Nerve Growth Factor Levels in the Synovial Fluid of Patients with Inflammatory Joint Disease

A novel pH shock extraction procedure was used to measure nerve growth factor (NGF) levels in both normal and inflamed synovial fluids using a sensitive and specific two-site enzyme linked immunosorbant assay. To date no data is available on NGF levels in normal synovial fluids. Synovial fluids were taken from 5 normal volunteers, 12 patients with rheumatoid arthritis and 10 patients with other inflammatory arthropathies. The mean ± SEM NGF concentration in normal synovial fluids was 95 ± 33.2 pg/ml (range 39.1–143.1 pg/ml), whereas the mean NGF concentration in the synovial fluids taken from patients with rheumatoid arthritis was 532.5 ± 123.8 pg/ml (range 152–1686 pg/ml). The mean NGF concentration in patients with other inflammatory arthropathies was also raised (430.6 ± 90 pg/ml; range 89–1071 pg/ml). The NGF concentrations were significantly higher in the synovial fluids from both inflamed groups (ANOVA p < 0.05) compared to normals. Raised levels of NGF in synovial fluid may contribute directly to joint inflammation via activation of inflammatory cells.     

Prorenin-renin axis in synovial fluid in patients with rheumatoid arthritis and osteoarthritis.

This study was undertaken 1) to determine whether or not renin is present in synovial fluid in patients with rheumatoid arthritis and osteoarthritis, and, if present, 2) to investigate whether it is synthesized in synovial fluid, or it is only transported from the circulation into the synovial cavity. The active renin concentration (indirect) was measured with angiotensin I radioimmunoassay kits. Inactive renin was converted into active renin with Sepharose-bound trypsin. Both active and inactive forms of renin were found in synovial fluid. They were significantly higher in patients with rheumatoid arthritis (n = 9) than in those with osteoarthritis (n = 16). In plasma, the concentration of inactive renin was significantly higher (P less than 0.001) in the former. Albumin, transferrin, alpha 2-macroglobulin, ceruloplasmin and immunoglobulins G and M were also found in synovial fluid. In each disease, a plot of the log ratio of synovial fluid to the serum concentration against the log molecular weight of each protein gave an approximately straight line curve, suggesting that these proteins are derived from the circulation and are transported into the synovial cavity. In contrast, the ratio of synovial fluid to plasma concentrations of active renin was significantly higher than that predicted on the basis of the above-mentioned interrelationships in both diseases, whereas the ratio of inactive renin was significantly lower. These findings suggest that 1) inactive and active renin are filtered into the synovial fluid from the circulation, and that 2) inactive renin is converted into the active form in the fluid.     

Differential synthesis of 5-lipoxygenase in peripheral blood and synovial fluid neutrophils in rheumatoid arthritis

In order to investigate 5-lipoxygenase enzyme regulation in neutrophils during an inflammatory reaction, we studied 5-lipoxygenase mRNA levels, as well as de novo enzyme synthesis, in resting and activated neutrophils isolated from normal individuals and patients with rheumatoid arthritis. The approach used was to analyze these activities in resting peripheral blood neutrophils of normal individuals on the one hand and in peripheral blood and matched synovial fluid neutrophils isolated from patients with rheumatoid arthritis on the other hand. Our first observation was that resting peripheral blood neutrophils of either normal individuals or patients show detectable levels of 5-lipoxygenase mRNA and are able to synthesize the enzyme de novo. Our second observation was that inflammatory activated neutrophils from synovial fluid reveal lower 5-lipoxygenase mRNA levels and enzyme synthesis than do the patient-matched peripheral blood cells. This is in spite of the fact that, for other proteins, synovial fluid neutrophils are equally or more active than their peripheral blood counterparts. We conclude that peripheral blood neutrophils are capable of synthesizing the enzyme, thus ensuring the turnover of the protein. Furthermore, complex regulatory mechanisms appear to take place in response to inflammation as it occurs in synovial fluids of patients with rheumatoid arthritis, leading to decreased mRNA levels and enzyme synthesis. Possible mechanisms of regulation are discussed and are presently under investigation.     

Rheumatoid synovial CD4+ T cells exhibit a reduced capacity to differentiate into IL-4-producing T-helper-2 effector cells

CD4+ memory T cells (Tm) from rheumatoid arthritis peripheral blood (RAPB) or peripheral blood from normal donors produced IL-2, whereas fewer cells secreted IFN-gamma or IL-4 after a brief stimulation. RAPB Tm contained significantly more IFN-gamma producers than normal cells. Many rheumatoid arthritis (RA) synovial Tm produced IFN-gamma alone (40%) and fewer cells produced IL-2 or IL-4. An in vitro model was employed to generate polarized T-helper (Th) effectors. Normal and RAPB Tm differentiated into both IFN-gamma- and IL-4-producing effectors. RA synovial fluid (RASF) Tm demonstrated defective responsiveness, exhibiting diminished differentiation of IL-4 effectors, whereas RA synovial tissue (RAST) Tm exhibited defective generation of IFN-gamma and IL-4 producers.     

Nitric Oxide in Arthritis

Nitric oxide’s (NO) involvement in arthritis was first demonstrated when levels of nitrite, a stable endproduct of NO metabolism, were shown to be elevated in serum and synovial fluid samples of rheumatoid and osteoarthritis patients. NO production by chondrocytes, its involvement in various biochemical events of cartilage metabolism, and the in vivo suppression of experimental arthritis by NO synthase inhibitors further implicated NO in arthritis. However, a conclusive role for NO in the pathogenesis of arthritis remains to be defined, in contrast to the NO-cGMP signal transduction pathway of endothelium-mediated vasodilation. It appears that NO has limited modulating effects in cartilage metabolism, with evidence for both protective and deleterious effects. Recent developments that contribute to our understanding of NO’s role in arthritis are discussed.     

The expression of IL-20 and IL-24 and their shared receptors are increased in rheumatoid arthritis and spondyloarthropathy

The purpose of this study was to analyze the expression of the two proinflammatory cytokines IL-20 and IL-24 and their shared receptors in rheumatoid arthritis and spondyloarthropathy. IL-20 was increased in plasma of rheumatoid arthritis patients compared with osteoarthritis patients and IL-24 was increased in synovial fluid and plasma of rheumatoid arthritis and spondyloarthropathy patients compared with osteoarthritis patients. IL-20 and IL-24 mRNA was only present at low levels in the synovium. In the synovial membrane, IL-20 protein was present in mononuclear cells and neutrophil granulocytes whereas IL-24 protein was observed in endothelial cells and mononuclear cells. IL-20 receptor type 1 and IL-22 receptor were expressed by granulocytes in the synovial fluid. In synovial fluid mononuclear cell cultures, stimulation with recombinant human IL-20 or recombinant human IL-24 induced monocyte chemoattractant protein 1 (CCL2/MCP-1) secretion, but not tumour necrosis factor alpha mRNA synthesis or IL-6 secretion. Both IL-20 and IL-24 showed correlations to CCL2/MCP-1 in plasma from rheumatoid arthritis and spondyloarthropathy patients. This study associates IL-20 and IL-24 to the synovium of rheumatoid arthritis and spondyloarthropathy and results indicate that the two cytokines contribute to disease pathogenesis through recruitment of neutrophil granulocytes and induction of CCL2/MCP-1.     

The role of Salmonella and Yersinia in the pathogenesis of spondyloarthropathies and rheumatoid arthritis. I. Detection of bacteria, bacterial DNA, lipopolysaccharide and ECA antigen in synovial fluid or blood

To investigate the role of Salmonella and Yersinia in the pathogenesis of spondyloarthropathies and rheumatoid arthritis synovial specimens from 92 patients were analysed for the presence of bacterial DNA with the use of polymerase chain reaction and for the presence of lipopolysaccharide and enterobacterial common antigen (ECA) with the use of Dot-ELISA. In addition, peripheral blood samples were available for PCR analysis from 68 patients. Salmonella and Yersinia chromosomal DNA was not found in any of the synovial specimens and blood samples from the patients. All of the synovial fluids were also culture-negative. Salmonella LPS antigens were observed in 8 (8.6%), Yersinia in 20 (21.7%) and ECA antigens in 32 (34.9%) synovial specimens. Our findings revealed the presence of bacterial degradation products, but not bacteria from the genus Salmonella and Yersinia or their DNA in the synovial fluid or blood of patients with spondyloarthropathies and rheumatoid arthritis.     

Measurement of sulfidopeptide leukotrienes and their metabolism in human synovial fluid of patients with rheumatoid arthritis

Leukotriene (LT)C4 in the synovial fluid of patients with osteoarthritis deformans (OA) and rheumatoid arthritis (RA) was measured by radioimmunoassay (RIA) after extraction with Sep-Pak C18 cartridge. The amounts of immunoreactive LTC4 (i-LTC4) in samples from patients with OA and RA were not significantly different, being 0.198 +/- 0.018 pmol/ml (n = 11) and 0.179 +/- 0.016 pmol/ml (n = 12), respectively. After separation by high performance liquid chromatography (HPLC) and measurement by RIA, the levels of other sulfidopeptide LTs, such as LTD4 and LTE4, in synovial fluid from patients with RA were found to be significantly higher than those in fluid from patients with OA. The leukocyte number in synovial fluids did not correlate with the i-LTC4 level. The metabolic activities of these synovial fluids were determined by incubating them with 3H-LTC4 and then separating sulfidopeptide LTs by HPLC. The conversion of LTC4 to LTD4 in synovial fluids of patients with OA and RA were similar, but the dipeptidase activity converting LTD4 to LTE4 was higher in fluid from patients with RA. It is suggested that a high level of LTE4 may contribute to exudation of synovial fluid, since LTE4 increases vascular permeability.     

The migratory and phagocytic activity of polymorphonuclear leukocytes in rheumatoid arthritis and osteoarthritis patients

Polymorphonuclear neutrophils (PMN) play a central role in the elimination of most extracellular pathogenic microorganisms and any impairment of their functions therefore predisposes to defect immune defence. We investigated the migratory and phagocytic functions of the PMNs isolated from peripheral blood and synovial fluid of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). The results suggest that in RA the number and the migratory but not phagocytic capacity of synovial fluid (SF) neutrophils were enhanced, while in OA they were significantly decreased in synovial fluid cells comparatively with peripheral blood (PB). The migratory function of both PB and SF cells from RA patients was increased comparatively with that of the cells from OA patients. We found the different abnormal functions in synovial fluid neutrophils from RA and OA patients. These results may help to elucidate the underlying mechanism which leads to severe joint destruction and different susceptibility to infectious diseases in patients with rheumatic disorders.     

Effects of synovial fluid on the respiratory burst of granulocytes in rheumatoid arthritis

Neutrophil infiltration in the synovia is an important feature of the local inflammatory process associated with rheumatoid arthritis. The present study is focused on the effects exerted in vitro by the synovial fluid versus serum on the respiratory burst of granulocytes isolated either from blood or synovial fluid of rheumatoid arthritis patients. The respiratory burst was evaluated as superoxide anion release, by lucigenin-amplified chemiluminescence. Our data show that the respiratory burst of granulocytes isolated from rheumatoid arthritis patients might trigger a significant oxidative stress both in periphery and the inflamed joint. These cells show no pathological pattern when activated in vitro by the chemotactic peptide fMLP, heterologous synovial fluid or serum. Acellular synovial fluid amplifies the superoxide anion release induced by fMLP more than the corresponding serum, indicating that a bacterian infection in the joint might enhance the oxidative damage in the inflamed synovium.     

Skewed distribution of proinflammatory CD4+CD28null T cells in rheumatoid arthritis

Expanded populations of CD4+ T cells lacking the co-stimulatory molecule CD28 (CD4+CD28null T cells) have been reported in several inflammatory disorders. In rheumatoid arthritis, increased frequencies of CD4+CD28null T cells in peripheral blood have previously been associated with extra-articular manifestations and human cytomegalovirus (HCMV) infection, but their presence in and contribution to joint manifestations is not clear. In the present article we investigated the distribution of CD4+CD28null T cells in the synovial membrane, synovial fluid and peripheral blood of RA patients, and analysed the association with erosive disease and anti-citrullinated protein antibodies. CD4+CD28null T cells were infrequent in the synovial membrane and synovial fluid, despite significant frequencies in the circulation. Strikingly, the dominant TCR-Vbeta subsets of CD4+CD28null T cells in peripheral blood were often absent in synovial fluid. CD4+CD28null T cells in blood and synovial fluid showed specificity for HCMV antigens, and their presence was clearly associated with HCMV seropositivity but not with anti-citrullinated protein antibodies in the serum or synovial fluid, nor with erosive disease. Together these data imply a primary role for CD4+CD28null T cells in manifestations elsewhere than in the joints of patients with HCMV-seropositive rheumatoid arthritis.     

Expression of MDC/CCL22 and its receptor CCR4 in rheumatoid arthritis,psoriatic arthritis and osteoarthritis

The pathogenesis of rheumatoid arthritis (RA) and psoriatic arthritis (PsA) involves an abnormal chemokine regulation. The chemokine receptor CCR4 is necessary for T cell migration to the skin. We, therefore, studied if CCR4 and its ligand macrophage-derived chemokine (MDC/CCL22) could participate in spreading the disease between skin and joints by examining RA, PsA and osteoarthritis (OA) patients. In synovial fluid from RA and PsA patients we observed a significantly higher MDC/CCL22 level compared to OA patients. Additionally, the MDC/CCL22 protein was found to be elevated in RA and PsA plasma compared to OA and healthy volunteers. Flow cytometry revealed that most CD4⁺CCR4⁺ lymphocytes also co-expressed CD45RO. Neither the MDC/CCL22 level nor the expression of CCR4 correlated to CRP. Immunohistochemistry of the RA and OA synovial membrane demonstrated CCR4 to be expressed by mononuclear cells and endothelial cells. Our results show that MDC/CCL22 is present within the synovial membrane of RA and OA patients and in high amount in the synovial fluid of patients with RA and PsA. This will enable migration of CCR4 expressing memory cells supporting that MDC/CCR4 could play a role in attracting skin specific memory T cells to the joints.