Chemokine secretion of rheumatoid arthritis synovial fibroblasts stimulated by Toll-like receptor 2 ligands

To analyze the role of Toll-like receptors (TLR) in the pathogenesis of rheumatoid arthritis, we have assessed the effects of stimulation of cultured synovial fibroblasts by the TLR-2 ligand bacterial peptidoglycan. By using high density oligonucleotide microarray analysis we identified 74 genes that were up-regulated >2.5-fold. Fourteen CC and CXC chemokine genes were among the genes with the highest up-regulation. Quantitative real-time PCR analysis confirmed up-regulation of granulocyte chemotactic protein (GCP)-2, RANTES, monocyte chemoattractant protein (MCP)-2, IL-8, growth-related oncogene-2, and to a lesser extent, macrophage-inflammatory protein 1alpha, MCP-1, EXODUS, and CXCL-16. GCP-2, RANTES, and MCP-2 were detected in culture supernatants of synovial fibroblasts stimulated with peptidoglycan. Chemokine secretion induced by stimulation of rheumatoid arthritis synovial fibroblasts via TLR-2 was functionally relevant as demonstrated by chemotaxis assays. GCP-2 and MCP-2 expression, which have not been reported previously in rheumatoid arthritis, was demonstrated in synovial tissue sections of patients diagnosed with rheumatoid arthritis but not in those with osteoarthritis. Correspondingly, synovial fluid levels were significantly higher in patients diagnosed with rheumatoid arthritis as compared with osteoarthritis. Thus, we present evidence for an induction of chemokine secretion by activation of synovial fibroblasts via TLR-2, possibly contributing to the formation of inflammatory infiltrates characteristically found in rheumatoid arthritis joints.     

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.     

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.     

Interleukin-18 induces serum amyloid A (SAA) protein production from rheumatoid synovial fibroblasts

Interleukin-18 (IL-18) is a novel proinflammatory cytokine that was recently found in synovial fluids and synovial tissues from patients with rheumatoid arthritis (RA). To investigate the role of IL-18 in rheumatoid synovitis, the levels of IL-18 and serum amyloid A (SAA) were measured in synovial fluids from 24 patients with rheumatoid arthritis (RA) and 13 patients with osteoarthritis (OA). The levels of IL-18 and SAA in the synovial fluids were elevated in RA patients. In contrast, the levels of IL-18 in synovial fluids from OA patients were significantly lower compared to those of RA patients. SAA was not detected in synovial fluids from OA patients. The expression of SAA mRNA in rheumatoid synovial cells was also examined. SAA4 mRNA, which was constitutively expressed by rheumatoid synovial cells, was not affected by IL-18 stimulation. Although acute phase SAA (A-SAA, SAA1 + 2) mRNA was not detected in unstimulated synovial cells, its expression was induced by IL-18 stimulation. By immunoblot, we demonstrated that IL-18 induced the SAA protein synthesis from rheumatoid synovial cells in a dose-dependent manner. These results indicate a novel role for IL-18 in rheumatoid inflammation through the synovial SAA production.     

The identification and characterization of a novel protein,c19orf10, in the synovium

Joint inflammation and destruction have been linked to the deregulation of the highly synthetic fibroblast-like synoviocytes (FLSs), and much of our current understanding of the mechanisms that underlie synovitis has been collected from studies of FLSs. During a proteomic analysis of FLS cells, we identified a novel protein, c19orf10 (chromosome 19 open reading frame 10), that was produced in significant amounts by these cells. The present study provides a partial characterization of c19orf10 in FLSs, synovial fluid, and the synovium. Murine monoclonal and chicken polyclonal antibodies were produced against recombinant human c19orf10 protein and used to examine the distribution of c19orf10 in cultured FLSs and in synovial tissue sections from patients with rheumatoid arthritis or osteoarthritis. The intracellular staining pattern of c19orf10 is consistent with localization in the endoplasmic reticulum/Golgi distribution. Sections of rheumatoid arthritis and osteoarthritis synovia expressed similar patterns of c19orf10 distribution with perivascular and synovial lining staining. Double-staining in situ analysis suggests that fibroblast-like synovial cells produced c19orf10, whereas macrophages, B cells, or T cells produced little or none of this protein. There is evidence of secretion into the vascular space and the extracellular matrix surrounding the synovial lining. A competitive enzyme-linked immunosorbent assay confirmed the presence of microgram levels of c19orf10 in the synovial fluids of patients with one of various arthropathies. Collectively, these results suggest that c19orf10 is an FLS-derived protein that is secreted into the synovial fluid. However, the significance of this protein in synovial biology remains to be determined. The absence of known structural motifs or domains and its relatively late evolutionary appearance raise interesting questions about its function.     

Identification of small heat shock protein B8 (HSP22) as a novel TLR4 ligand and potential involvement in the pathogenesis of rheumatoid arthritis

Dendritic cells (DCs) are specialized APCs that can be activated upon pathogen recognition as well as recognition of endogenous ligands, which are released during inflammation and cell stress. The recognition of exogenous and endogenous ligands depends on TLRs, which are abundantly expressed in synovial tissue from rheumatoid arthritis (RA) patients. Furthermore TLR ligands are found to be present in RA serum and synovial fluid and are significantly increased, compared with serum and synovial fluid from healthy volunteers and patients with systemic sclerosis and systemic lupus erythematosus. Identification of novel endogenous TLR ligands might contribute to the elucidation of the role of TLRs in RA and other autoimmune diseases. In this study, we investigated whether five members of the small heat shock protein (HSP) family were involved in TLR4-mediated DC activation and whether these small HSPs were present in RA synovial tissue. In vitro, monocyte-derived DCs were stimulated with recombinant alphaA crystallin, alphaB crystallin, HSP20, HSPB8, and HSP27. Using flow cytometry and multiplex cytokine assays, we showed that both alphaA crystallin and HSPB8 were able to activate DCs and that this activation was TLR4 dependent. Furthermore, Western blot and immunohistochemistry showed that HSPB8 was abundantly expressed in synovial tissue from patients with RA. With these experiments, we identified sHSP alphaA crystallin and HSPB8 as two new endogenous TLR4 ligands from which HSPB8 is abundantly expressed in RA synovial tissue. These findings suggest a role for HSPB8 during the inflammatory process in autoimmune diseases such as RA.     

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.     

Characteristics of tumor necrosis factor production in rheumatoid arthritis

The biological effects of tumor necrosis factor (TNF) include the enhancement of fibroblast proliferation, the secretion of collagenase and prostaglandin E2 (PGE2) by fibroblasts, and the resorption of bone and cartilage, suggesting a role for this cytokine in arthritic conditions. To investigate this, we measured the levels of TNF in synovial fluids and evaluated its secretion by synovial fluid mononuclear cells and tissues from patients with rheumatoid arthritis, osteoarthritis, and seronegative arthritis and normals. TNF was found to be secreted in all arthritic conditions but not in normals. The levels of TNF were highest in synovial fluid and correlated with interferon-gamma (IFN-gamma) levels but not PGE2. The production of TNF was stable in a single joint for 3 to 6 months. Using immunohistochemical staining, TNF was localized to mononuclear cells in the lining layer, sublining, and perivascular areas of synovial tissue. The secretion of TNF by rheumatoid synovial fluid mononuclear cells was inhibited by PGE2, while IFN-gamma enhanced its production in those cells which were spontaneously secreting TNF. Our data suggest that TNF may play a role in various arthritic diseases.     

Interleukin-1 induced gene expression of neutrophil activating protein (interleukin-8) and monocyte chemotactic peptide in human synovial cells

We report here that human synovial cells stimulated by interleukin-1 alpha and interleukin-1 beta express mRNA for both IL-8 (neutrophil chemotactic peptide) and monocyte chemotactic protein. IL-1 stimulated synovial cells from both osteoarthritis and rheumatoid arthritis patients exhibited similar mRNA expression of interleukin-8 and monocyte chemotactic protein. A capacity to produce factors selectively chemotactic for neutrophils, lymphocytes and monocytes provides a mechanism whereby synovial cells can facilitate inflammatory arthritis.     

The metabolism of synthetic leukotriene B4 in synovial fluid and whole human blood

The metabolism in vitro 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 than 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 in vivo will in part determine the available concentration of LTB4 in inflammatory lesions.     

Extracellular heat shock protein 70 inhibits tumour necrosis factor-α induced proinflammatory mediator production in fibroblast-like synoviocytes

Introduction  

It was recently suggested that heat shock protein (HSP)70, an intracellular protein, is a potential mediator of inflammatory disease when it is released into the extracellular compartment. Although elevated HSP70 levels have been identified in rheumatoid arthritis (RA) synovial tissues and RA synovial fluid compared with patients with osteoarthritis and healthy individuals, it remains unclear what role extracellular HSP70 plays in the pathogenesis of RA. This study was conducted to investigate the effects of extracellular HSP70 on the production of RA-associated cytokines in fibroblast-like synoviocytes from patients with RA and to elucidate the mechanisms involved.     

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.     

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.     

Pro-inflammatory cytokine production by synoviocytes following exposure to protein I/II, a modulin from oral streptococci

We have tested the ability of protein I/II, an adhesin from oral streptococci, to stimulate the production of pro-inflammatory cytokines by synovial cells isolated from both rheumatoid arthritis and control patients. Protein I/II triggers synovial fluid cells to produce interleukin (IL)-6 and IL-8 while secretion of tumor necrosis factor-alpha (TNF-alpha) was less enhanced. Using fibroblast-like synoviocytes, we found that protein I/II also exerts an immunomodulatory effect (IL-6 and IL-8 release) on these cells. These findings indicate that, if it gains access to the joint cavity, protein I/II could participate in the initiation and/or perpetuation of rheumatic diseases, by stimulating pro-inflammatory cytokine release from various synovial cells.     

Decreased levels of serum platelet-activating factor acetylhydrolase in patients with rheumatic diseases

PAF is a potent inflammatory compound known to stimulate the release of various cytokines involved in rheumatic diseases. Elevated blood PAF levels are reported in these patients. We report that serum PAF acetylhydrolase activity (AHA) levels are decreased in patients with rheumatoid arthritis or osteoarthritis as compared to healthy controls. Serum and synovial fluid AHA levels were correlated in these patients. The present study suggests the potential role of AHA in controling systemic and/or local PAF levels in patients with rheumatic diseases.     

A possible role for TSLP in inflammatory arthritis

Thymic stromal lymphopoietin (TSLP) is an IL-7-like cytokine that triggers dendritic cell-mediated Th2-type inflammatory responses and is considered as a master switch for allergic inflammation. In this study, we found increased levels of TSLP and, also TNF-alpha as previously reported, in synovial fluid specimens derived from patients with rheumatoid arthritis (RA) when compared with those from patients with osteoarthritis (OA). In addition, TNF-alpha up-regulated TSLP expression in RA- and OA-derived synovial fibroblasts, which was inhibited by IFN-gamma. Furthermore, anti-TSLP neutralizing antibody ameliorated a TNF-alpha-dependent experimental arthritis induced by anti-type II collagen antibody in mice. Collectively, these results suggest that TSLP, as a downstream molecule of TNF-alpha, may be involved in the pathophysiology of inflammatory arthritis. TSLP might thus play a role not only in allergic diseases but also in inflammatory arthritis such as RA.     

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.     

Synovial tissue macrophage as a source of the chemotactic cytokine IL-8

Cells of the synovial microenvironment may recruit neutrophils (PMN) and lymphocytes into synovial fluid, as well as lymphocytes into the synovial tissues, of arthritic patients. We have investigated the production of the chemotactic cytokine IL-8 by using sera, synovial fluid, synovial tissue, and macrophages and fibroblasts isolated from synovial tissues from 75 arthritic patients. IL-8 levels were higher in synovial fluid from rheumatoid (RA) patients (mean +/- SE, 14.37 +/- 5.8 ng/ml), compared with synovial fluid from osteoarthritis patients (0.135 +/- 17 ng/ml) (p less than 0.05) or from patients with other arthritides (5.52 +/- 5.11 ng/ml). IL-8 from RA sera was 8.44 +/- 2.33 ng/ml, compared with nondetectable levels found in normal sera. IL-8 levels from RA sera and synovial fluid were strongly positively correlated (r = 0.96, p less than 0.05). Moreover, RA synovial fluid chemotactic activity for PMN in these fluids was inhibited 40 +/- 5% upon incubation with neutralizing polyclonal antibody to IL-8. Synovial tissue fibroblasts released only small amounts of constitutive IL-8 but could be induced to produce IL-8 by stimulation with either IL-1 beta, TNF-alpha, or LPS. In contrast, unlike normal PBMC or alveolar macrophages, macrophages isolated from RA synovial tissue constitutively expressed both IL-8 mRNA and antigenic IL-8. RA synovial macrophage IL-8 expression was not augmented by incubation with either LPS, TNF-alpha, or IL-1 beta. Immunohistochemical analysis of synovial tissue showed that a greater percentage of RA macrophages than osteoarthritis macrophages reacted with anti-IL-8. Whereas macrophages were the predominant cell for immunolocalization of IL-8, less than 5% of synovial tissue fibroblasts were positive for immunolocalized IL-8. These results suggest that macrophage-derived IL-8 may play an important role in the recruitment of PMN in synovial inflammation associated with RA.     

α-Enolase expressed on the surfaces of monocytes and macrophages induces robust synovial inflammation in rheumatoid arthritis

α-Enolase (ENO1) is a multifunctional glycolytic enzyme expressed abundantly in the cytosol. It has been implicated in autoimmune and inflammatory diseases. Serum Abs against ENO1 were reported in rheumatoid arthritis (RA). Cell-surface expression of ENO1 has been found to be increased rapidly in response to inflammatory stimuli, but its expression and function has not been reported in RA. In this study, we show that cell-surface expression of ENO1 is increased on monocytes and macrophages isolated from RA patients but not on those from osteoarthritis patients, and Ab against ENO1 can stimulate these cells to produce higher amounts of proinflammatory mediators, such as TNF-α, IL-1 α/β, IFN-γ, and PGE(2) via p38 MAPK and NF-κB pathway. The frequency of ENO1-positive cells in synovial fluid mononuclear cells was higher than PBMCs. ENO1-positive cells were also found in the inflamed synovium from RA patients and arthritic ankle tissues of mice with collagen-induced arthritis. Taken together, these findings suggest that Abs against ENO1 present in RA sera may stimulate monocytes and macrophages expressing cell-surface ENO1 and contribute to production of proinflammatory mediators during the effector phase of synovial inflammation.