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1.  Mast cell activation and its relation to proinflammatory cytokine production in the rheumatoid lesion 
Arthritis Research  1999;2(1):65-74.
Mast cell (MC) activation in the rheumatoid lesion provides numerous mediators that contribute to inflammatory and degradative processes, especially at sites of cartilage erosion. MC activation in rheumatoid synovial tissue has often been associated with tumour necrosis factor (TNF)-α and interleukin (IL)-1β production by adjacent cell types. By contrast, our in situ and in vitro studies have shown that the production of IL-15 was independent of MC activation, and was not related to TNF-α and IL-1β expression. Primary cultures of dissociated rheumatoid synovial cells produced all three proinflammatory cytokines, with production of IL-1β exceeding that of TNF-α, which in turn exceeded that of IL-15. In vitro cultures of synovial macrophages, synovial fibroblasts and articular chondrocytes all produced detectable amounts of free IL-15, macrophages being the most effective.
Introduction:
Increased numbers of mast cells (MCs) are found in the synovial tissues and fluids of patients with rheumatoid arthritis (RA), and at sites of cartilage erosion. MC activation has been reported for a significant proportion of rheumatoid specimens. Because the MC contains potent mediators, including histamine, heparin, proteinases, leukotrienes and multifunctional cytokines, its potential contributions to the processes of inflammation and matrix degradation have recently become evident.
Proinflammatory cytokines are important mediators of inflammation, immunity, proteolysis, cell recruitment and proliferation. Tumour necrosis factor (TNF) reportedly plays a pivotal role in the pathogenesis of RA, especially its ability to regulate interleukin (IL)-1β expression, this being important for the induction of prostanoid and matrix metalloproteinase production by synovial fibroblasts and chondrocytes. IL-15 has been assigned numerous biological effects and has been implicated as an important factor in TNF-α expression by monocyte/macrophages. Some in vitro studies have placed IL-15 upstream from TNF-α in the cytokine cascade, suggesting an interdependence between TNF, IL-1 and IL-15 for the promotion of proinflammatory cytokine expression in the rheumatoid joint.
Aims:
To examine the in situ relationships of TNF-α, IL-1β and IL-15 in relation to MC activation in rheumatoid tissues by use of immunolocalization techniques; and to compare quantitatively the proinflammatory cytokine production by specific cell cultures and rheumatoid synovial explants with and without exposure to a MC secretagogue.
Materials and methods:
Samples of rheumatoid synovial tissue and cartilage–pannus junction were obtained from patients (n = 15) with classic late-stage RA. Tissue sections were immunostained for MC (tryptase) and the proinflammatory cytokines IL-1, TNF-α and IL-15. Rheumatoid synovial tissue explants were cultured in Dulbecco's modified Eagles medium (DMEM) containing either the MC secretagogue rabbit antihuman immunoglobulin (Ig)E, or control rabbit IgG. Primary rheumatoid synovial cell cultures, human articular chondrocytes, synovial fibroblasts and synovial macrophages were prepared as described in the full article. Conditioned culture media from these cultures were collected and assayed for IL-1β, TNF-α and IL-15 using enzyme-linked immunosorbent assay methodology.
Results:
Immunohistological studies of rheumatoid synovial tissues have demonstrated local concentrations of MCs in most specimens of the rheumatoid lesion. Sites of MC activation were associated with localized oedema, and TNF-α, IL-1α and IL-1β production by a proportion of mononuclear inflammatory cells. By contrast, no evidence was found for IL-15 production in tissue sites containing either intact or activated MCs, and IL-15 expression, when observed, bore no relation to tissue sites where TNF-α and IL-1β were evident. The immunodetection of IL-15 was restricted to microfocal sites and was not typical of most junctional specimens, but was associated with a proportion of articular chondrocytes in a minority of junctional specimens.
MC activation within synovial explant cultures was induced by the addition of polyclonal antibody to human IgE. MC activation significantly reduced the levels of TNF-α and IL1β released into the medium, this representing approximately 33% of control values. By contrast, MC activation had little effect on the levels of IL-15 released into the culture medium, the average value being very low in relation to the release of TNF-α and IL-1β . Thus, induced MC activation brings about changes in the amounts of released tryptase, TNF-α and IL-1β , but not of IL-15.
Four preparations of primary rheumatoid synovial cell cultures produced more IL-1β than TNF-α, with only modest values for IL-15 production, indicating that all three cytokines are produced and released as free ligands by these cultures. Of specific cell types that produced IL-15 in vitro, macrophages produced more than fibroblasts, which in turn produced more than chondrocytes. This demonstrates that all three cell types have the potential to produce IL-15 in situ.
Discussion:
The biological consequences of MC activation in vivo are extremely complex, and in all probability relate to the release of various combinations of soluble and granular factors, as well as to the expression of appropriate receptors by neighbouring cells. The subsequent synthesis and release of cytokines such as TNF-α and IL-1 may well follow at specific stages after activation, or may be an induced cytokine response by adjacent macrophagic or fibroblastic cells. However, because no IL-15 was detectable either in or around activated or intact MCs, and the induced MC activation explant study showed no change in IL-15 production, it seems unlikely that the expression of this cytokine is regulated by MCs. The immunohistochemistry (IHC) demonstration of IL-15 at sites of cartilage erosion, and especially by some chondrocytes of articular cartilage, showed no spatial relationship with either T cells or neutrophils, and suggests other functional properties in these locations. The lack of evidence for an in situ association of IL-15 with TNF and IL-1 does not support a role for IL-15 in a proinflammatory cytokine 'cascade', as proposed by other in vitro experiments. We believe that sufficient evidence is available, however, to suggest that MC activation makes a significant contribution to the pathophysiological processes of the rheumatoid lesion.
PMCID: PMC17805  PMID: 11219391
interleukin-15; interleukin-1β; mast cells; rheumatoid arthritis; tumour necrosis factor-α
2.  Blockade of Toll-like receptor 2 prevents spontaneous cytokine release from rheumatoid arthritis ex vivo synovial explant cultures 
Introduction
The aim of this study was to examine the effect of blocking Toll-like receptor 2 (TLR2) in rheumatoid arthritis (RA) synovial cells.
Methods
RA synovial tissue biopsies, obtained under direct visualization at arthroscopy, were established as synovial explant cultures ex vivo or snap frozen for immunohistology. Mononuclear cell cultures were isolated from peripheral blood and synovial fluid of RA patients. Cultures were incubated with the TLR1/2 ligand, Pam3CSK4 (200 ng, 1 and 10 μg/ml), an anti-TLR2 antibody (OPN301, 1 μg/ml) or an immunoglobulin G (IgG) (1 μg/ml) matched control. The comparative effect of OPN301 and adalimumab (anti-tumour necrosis factor alpha) on spontaneous release of proinflammatory cytokines from RA synovial explants was determined using quantitative cytokine MSD multiplex assays or ELISA. OPN301 penetration into RA synovial tissue explants cultures was assessed by immunohistology.
Results
Pam3CSK4 significantly upregulated interleukin (IL)-6 and IL-8 in RA peripheral blood mononuclear cells (PBMCs), RA synovial fluid mononuclear cells (SFMCs) and RA synovial explant cultures (P < 0.05). OPN301 significantly decreased Pam3CSK4-induced cytokine production of tumour necrosis factor alpha (TNF-α), IL-1β, IL-6, interferon (IFN)-γ and IL-8 compared to IgG control in RA PBMCs and SFMCs cultures (all P < 0.05). OPN301 penetration of RA synovial tissue cultures was detected in the lining layer and perivascular regions. OPN301 significantly decreased spontaneous cytokine production of TNF-α, IL-1β, IFN-γ and IL-8 from RA synovial tissue explant cultures (all P < 0.05). Importantly, the inhibitory effect of OPN on spontaneous cytokine secretion was comparable to inhibition by anti-TNFα monoclonal antibody adalimumab.
Conclusions
These findings further support targeting TLR2 as a potential therapeutic agent for the treatment of RA.
doi:10.1186/ar3261
PMCID: PMC3241377  PMID: 21345222
3.  Acute-phase serum amyloid A production by rheumatoid arthritis synovial tissue 
Arthritis Research  2000;2(2):142-144.
Acute-phase serum amyloid A (A-SAA) is a major component of the acute-phase response. A sustained acute-phase response in rheumatoid arthritis (RA) is associated with increased joint damage. A-SAA mRNA expression was confirmed in all samples obtained from patients with RA, but not in normal synovium. A-SAA mRNA expression was also demonstrated in cultured RA synoviocytes. A-SAA protein was identified in the supernatants of primary synoviocyte cultures, and its expression colocalized with sites of macrophage accumulation and with some vascular endothelial cells. It is concluded that A-SAA is produced by inflamed RA synovial tissue. The known association between the acute-phase response and progressive joint damage may be the direct result of synovial A-SAA-induced effects on cartilage degradation.
Introduction:
Serum amyloid A (SAA) is the circulating precursor of amyloid A protein, the fibrillar component of amyloid deposits. In humans, four SAA genes have been described. Two genes (SAA1 and SAA2) encode A-SAA and are coordinately induced in response to inflammation. SAA1 and SAA2 are 95% homologous in both coding and noncoding regions. SAA3 is a pseudogene. SAA4 encodes constitutive SAA and is minimally inducible. A-SAA increases dramatically during acute inflammation and may reach levels that are 1000-fold greater than normal. A-SAA is mainly synthesized in the liver, but extrahepatic production has been demonstrated in many species, including humans. A-SAA mRNA is expressed in RA synoviocytes and in monocyte/macrophage cell lines such as THP-1 cells, in endothelial cells and in smooth muscle cells of atherosclerotic lesions. A-SAA has also been localized to a wide range of histologically normal tissues, including breast, stomach, intestine, pancreas, kidney, lung, tonsil, thyroid, pituitary, placenta, skin and brain.
Aims:
To identify the cell types that produce A-SAA mRNA and protein, and their location in RA synovium.
Materials and methods:
Rheumatoid synovial tissue was obtained from eight patients undergoing arthroscopic biopsy and at joint replacement surgery. Total RNA was analyzed by reverse transcription (RT) polymerase chain reaction (PCR) for A-SAA mRNA. PCR products generated were confirmed by Southern blot analysis using human A-SAA cDNA. Localization of A-SAA production was examined by immunohistochemistry using a rabbit antihuman A-SAA polyclonal antibody. PrimaryRA synoviocytes were cultured to examine endogenous A-SAA mRNA expression and protein production.
Results:
A-SAA mRNA expression was detected using RT-PCR in all eight synovial tissue samples studied. Figure 1 demonstrates RT-PCR products generated using synovial tissue from three representative RA patients. Analysis of RA synovial tissue revealed differences in A-SAA mRNA levels between individual RA patients.
In order to identify the cells that expressed A-SAA mRNA in RA synovial tissue, we analyzed primary human synoviocytes (n = 2). RT-PCR analysis revealed A-SAA mRNA expression in primary RA synoviocytes (n = 2; Fig. 2). The endogenous A-SAA mRNA levels detected in individual primary RA synoviocytes varied between patients. These findings are consistent with A-SAA expression in RA synovial tissue (Fig. 1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels were relatively similar in the RA synoviocytes examined (Fig. 2). A-SAA protein in the supernatants of primary synoviocyte cultures from four RA patients was measured using ELISA. Mean values of a control and four RA samples were 77.85, 162.5, 249.8, 321.5 and 339.04 μg/l A-SAA, respectively, confirming the production of A-SAA protein by the primary RA synoviocytes. Immunohistochemical analysis was performed to localize sites of A-SAA production in RA synovial tissue. Positive staining was present in both the lining and sublining layers of all eight RA tissues examined (Fig. 3a). Staining was intense and most prominent in the cells closest to the surface of the synovial lining layer. Positively stained cells were evident in the perivascular areas of the sublining layer. In serial sections stained with anti-CD68 monoclonal antibody, positive staining of macrophages appeared to colocalize with A-SAA-positive cells (Fig. 3b). Immunohistochemical studies of cultured primary RA synoviocytes confirmed specific cytoplasmic A-SAA expression in these cells. The specificity of the staining was confirmed by the absence of staining found on serial sections and synoviocyte cells treated with IgG (Fig. 3c).
Discussion:
This study demonstrates that A-SAA mRNA is expressed in several cell populations infiltrating RA synovial tissue. A-SAA mRNA expression was observed in all eight unseparated RA tissue samples studied. A-SAA mRNA expression and protein production was demonstrated in primary cultures of purified RA synoviocytes. Using immunohistochemical techniques, A-SAA protein appeared to colocalize with both lining layer and sublining layer synoviocytes, macrophages and some endothelial cells. The detection of A-SAA protein in culture media supernatants harvested from unstimulated synoviocytes confirms endogenous A-SAA production, and is consistent with A-SAA mRNA expression and translation by the same cells. Moreover, the demonstration of A-SAA protein in RA synovial tissue, RA cultured synoviocytes, macrophages and endothelial cells is consistent with previous studies that demonstrated A-SAA production by a variety of human cell populations.
The RA synovial lining layer is composed of activated macrophages and fibroblast-like synoviocytes. The macrophage is the predominant cell type and it has been shown to accumulate preferentially in the surface of the lining layer and in the perivascular areas of the sublining layer. Nevertheless, our observations strongly suggest that A-SAA is produced not only by synoviocytes, but also by synovial tissue macrophage populations. Local A-SAA protein production by vascular endothelial cells was detected in some, but not all, of the tissues examined. The reason for the variability in vascular A-SAA staining is unknown, but may be due to differences in endothelial cell activation, events related to angiogenesis or the intensity of local inflammation.
The value of measuring serum A-SAA levels as a reliable surrogate marker of inflammation has been demonstrated for several diseases including RA, juvenile chronic arthritis, psoriatic arthropathy, ankylosing spondylitis, Behçet's disease, reactive arthritis and Crohn's disease. It has been suggested that serum A-SAA levels may represent the most sensitive measurement of the acute-phase reaction. In RA, A-SAA levels provide the strongest correlations with clinical measurements of disease activity, and changes in serum levels best reflect the clinical course.
A number of biologic activities have been described for A-SAA, including several that are relevant to the understanding of inflammatory and tissue-degrading mechanisms in human arthritis. A-SAA induces migration, adhesion and tissue infiltration of circulating monocytes and polymorphonuclear leukocytes. In addition, human A-SAA can induce interleukin-1β, interleukin-1 receptor antagonist and soluble type II tumour necrosis factor receptor production by a monocyte cell line. Moreover, A-SAA can stimulate the production of cartilage-degrading proteases by both human and rabbit synoviocytes. The effects of A-SAA on protease production are interesting, because in RA a sustained acute-phase reaction has been strongly associated with progressive joint damage. The known association between the acute-phase response and progressive joint damage may be the direct result of synovial A-SAA-induced effects on cartilage degradation.
Conclusion:
In contrast to noninflamed synovium, A-SAA mRNA expression was identified in all RA tissues examined. A-SAA appeared to be produced by synovial tissue synoviocytes, macrophages and endothelial cells. The observation of A-SAA mRNA expression in cultured RA synoviocytes and human RA synovial tissue confirms and extends recently published findings that demonstrated A-SAA mRNA expression in stimulated RA synoviocytes, but not in unstimulated RA synoviocytes.
PMCID: PMC17807  PMID: 11062604
acute-phase response; rheumatoid arthritis; serum amyloid A; synovial tissue
4.  Epithelial neutrophil activating peptide-78: a novel chemotactic cytokine for neutrophils in arthritis. 
Journal of Clinical Investigation  1994;94(3):1012-1018.
We and others have shown that cells obtained from inflamed joints of rheumatoid arthritis (RA) patients produce interleukin-8, a potent chemotactic cytokine for neutrophils (PMNs). However, IL-8 accounted for only 40% of the chemotactic activity for PMNs found in these synovial fluids. Currently, we have examined the production of the novel PMN chemotactic cytokine, epithelial neutrophil activating peptide-78 (ENA-78), using peripheral blood, synovial fluid, and synovial tissue from 70 arthritic patients. RA ENA-78 levels were greater in RA synovial fluid (239 +/- 63 ng/ml) compared with synovial fluid from other forms of arthritis (130 +/- 118 ng/ml) or osteoarthritis (2.6 +/- 1.8 ng/ml) (P < 0.05). RA peripheral blood ENA-78 levels (70 +/- 26 ng/ml) were greater than normal peripheral blood levels (0.12 +/- 0.04 ng/ml) (P < 0.05). Anti-ENA-78 antibodies neutralized 42 +/- 9% (mean +/- SE) of the chemotactic activity for PMNs found in RA synovial fluids. Isolated RA synovial tissue fibroblasts in vitro constitutively produced significant levels of ENA-78, and this production was further augmented when stimulated with tumor necrosis factor-alpha (TNF-alpha). In addition RA and osteoarthritis synovial tissue fibroblasts as well as RA synovial tissue macrophages were found to constitutively produce ENA-78. RA synovial fluid mononuclear cells spontaneously produced ENA-78, which was augmented in the presence of lipopolysaccharide. Immunohistochemical localization of ENA-78 from the synovial tissue of patients with arthritis or normal subjects showed that the predominant cellular source of this chemokine was synovial lining cells, followed by macrophages, endothelial cells, and fibroblasts. Synovial tissue macrophages and fibroblasts were more ENA-78 immunopositive in RA than in normal synovial tissue (P < 0.05). These results, which are the first demonstration of ENA-78 in a human disease state, suggest that ENA-78 may play an important role in the recruitment of PMNs in the milieu of the inflamed joint of RA patients.
Images
PMCID: PMC295150  PMID: 8083342
5.  Enhanced production of monocyte chemoattractant protein-1 in rheumatoid arthritis. 
Journal of Clinical Investigation  1992;90(3):772-779.
Cells within the synovial tissue may recruit mononuclear phagocytes into the synovial fluid and tissues of arthritic patients. We investigated the production of the chemotactic cytokine monocyte chemoattractant protein-1 (MCP-1) using sera, synovial fluid, synovial tissue, as well as macrophages and fibroblasts isolated from synovial tissues from 80 arthritic patients. MCP-1 levels were significantly higher (P less than 0.05) in synovial fluid from RA patients (mean 25.5 +/- 8.1 ng/ml [SE]) compared to synovial fluid from osteoarthritis (OA) patients (0.92 +/- 0.08), or from patients with other arthritides (2.9 +/- 1.5). MCP-1 levels in RA sera (8.44 +/- 2.33) were significantly greater than MCP-1 in normal sera (0.16 +/- 0.06). The quantities of RA synovial fluid IL-8, which is chemotactic for neutrophils and lymphocytes, and MCP-1 were strongly positively correlated (P less than 0.05). To examine the cellular source of MCP-1, RA synovial tissue macrophages and fibroblasts were isolated. Synovial tissue fibroblasts did not express MCP-1 mRNA, but could be induced to produce MCP-1 by stimulation with either IL-1 beta, tumor necrosis factor-alpha (TNF-alpha), or LPS. In contrast, unlike normal peripheral blood monocytes or alveolar macrophages, RA synovial tissue macrophages constitutively expressed MCP-1 mRNA and antigen. Immunohistochemical analysis of synovial tissue showed that a significantly greater percentage of RA macrophages (50 +/- 8%) as compared to either OA macrophages (5 +/- 2) or normal macrophages (1 +/- 0.3) reacted with anti-MCP-1 antibodies. In addition, the synovial lining layer reacted with MCP-1 in both RA and OA synovial tissues. In contrast, only a minority of synovial fibroblasts (18 +/- 8%) from RA synovium were positive for immunolocalization of MCP-1. These results suggest that synovial production of MCP-1 may play an important role in the recruitment of mononuclear phagocytes during inflammation associated with RA and that synovial tissue macrophages are the dominant source of this cytokine.
Images
PMCID: PMC329929  PMID: 1522232
6.  Activation of synovial fibroblasts in rheumatoid arthritis: lack of expression of the tumour suppressor PTEN at sites of invasive growth and destruction 
Arthritis Research  1999;2(1):59-64.
In the present study, we searched for mutant PTEN transcripts in aggressive rheumatoid arthritis synovial fibroblasts (RA-SF) and studied the expression of PTEN in RA. By automated sequencing, no evidence for the presence of mutant PTEN transcripts was found. However, in situ hybridization on RA synovium revealed a distinct expression pattern of PTEN, with negligible staining in the lining layer but abundant expression in the sublining. Normal synovial tissue exhibited homogeneous staining for PTEN. In cultured RA-SF, only 40% expressed PTEN. Co-implantation of RA-SF and normal human cartilage into severe combined immunodeficiency (SCID) mice showed only limited expression of PTEN, with no staining in those cells aggressively invading the cartilage. Although PTEN is not genetically altered in RA, these findings suggest that a lack of PTEN expression may constitute a characteristic feature of activated RA-SF in the lining, and may thereby contribute to the invasive behaviour of RA-SF by maintaining their aggressive phenotype at sites of cartilage destruction.
Aims:
PTEN is a novel tumour suppressor which exhibits tyrosine phosphatase activity as well as homology to the cytoskeletal proteins tensin and auxilin. Mutations of PTEN have been described in several human cancers and associated with their invasiveness and metastatic properties. Although not malignant, rheumatoid arthritis synovial fibroblasts (RA-SF) exhibit certain tumour-like features such as attachment to cartilage and invasive growth. In the present study, we analyzed whether mutant transcripts of PTEN were present in RA-SF. In addition, we used in situ hybridization to study the expression of PTEN messenger (m)RNA in tissue samples of RA and normal individuals as well as in cultured RA-SF and in the severe combined immunodeficiency (SCID) mouse model of RA.
Methods:
Synovial tissue specimens were obtained from seven patients with RA and from two nonarthritic individuals. Total RNA was isolated from synovial fibroblasts and after first strand complementary (c)DNA synthesis, polymerase chain reaction (PCR) was performed to amplify a 1063 base pair PTEN fragment that encompassed the coding sequence of PTEN including the phosphatase domain and all mutation sites described so far. The PCR products were subcloned in Escherichia coli, and up to four clones were picked from each plate for automated sequencing. For in situ hybridization, digoxigenin-labelled PTEN-specific RNA probes were generated by in vitro transcription. For control in situ hybridization, a matrix metalloproteinase (MMP)-2-specific probe was prepared. To investigate the expression of PTEN in the absence of human macrophage or lymphocyte derived factors, we implanted RA-SF from three patients together with normal human cartilage under the renal capsule of SCID mice. After 60 days, mice were sacrificed, the implants removed and embedded into paraffin.
Results:
PCR revealed the presence of the expected 1063 base pair PTEN fragment in all (9/9) cell cultures (Fig. 1). No additional bands that could account for mutant PTEN variants were detected. Sequence analysis revealed 100% homology of all RA-derived PTEN fragments to those from normal SF as well as to the published GenBank sequence (accession number U93051). However, in situ hybridization demonstrated considerable differences in the expression of PTEN mRNA within the lining and the sublining layers of RA synovial membranes. As shown in Figure 2a, no staining was observed within the lining layer which has been demonstrated to mediate degradation of cartilage and bone in RA. In contrast, abundant expression of PTEN mRNA was found in the sublining of all RA synovial tissues (Figs 2a and b). Normal synovial specimens showed homogeneous staining for PTEN within the thin synovial membrane (Fig. 2c). In situ hybridization using the sense probe gave no specific staining (Fig. 2d). We also performed in situ hybridization on four of the seven cultured RA-SF and followed one cell line from the first to the sixth passage. Interestingly, only 40% of cultured RA-SF expressed PTEN mRNA (Fig. 3a), and the proportion of PTEN expressing cells did not change throughout the passages. In contrast, control experiments using a specific RNA probe for MMP-2 revealed mRNA expression by nearly all cultured cells (Fig. 3b). As seen before, implantation of RA-SF into the SCID mice showed considerable cartilage degradation. Interestingly, only negligible PTEN expression was found in those RA-SF aggressively invading the cartilage (Fig. 3c). In situ hybridization for MMP-2 showed abundant staining in these cells (Fig. 3d).
Discussion:
Although this study found no evidence for mutations of PTEN in RA synovium, the observation that PTEN expression is lacking in the lining layer of RA synovium as well as in more than half of cultured RA-SF is of interest. It suggests that loss of PTEN function may not exclusively be caused by genetic alterations, yet at the same time links the low expression of PTEN to a phenotype of cells that have been shown to invade cartilage aggressively.
It has been proposed that the tyrosine phosphatase activity of PTEN is responsible for its tumour suppressor activity by counteracting the actions of protein tyrosine kinases. As some studies have demonstrated an upregulation of tyrosine kinase activity in RA synovial cells, it might be speculated that the lack of PTEN expression in aggressive RA-SF contributes to the imbalance of tyrosine kinases and phosphatases in this disease. However, the extensive amino-terminal homology of the predicted protein to the cytoskeletal proteins tensin and auxilin suggests a complex regulatory function involving cellular adhesion molecules and phosphatase-mediated signalling. The tyrosine phosphatase TEP1 has been shown to be identical to the protein encoded by PTEN, and gene transcription of TEP1 has been demonstrated to be downregulated by transforming growth factor (TGF)-β. Therefore, it could be hypothesized that TGF-β might be responsible for the downregulation of PTEN. However, the expression of TGF-β is not restricted to the lining but found throughout the synovial tissue in RA. Moreover, in our study the percentage of PTEN expressing RA-SF remained stable for six passages in culture, whereas molecules that are cytokine-regulated in vivo frequently change their expression levels when cultured over several passages. Also, cultured RA-SF that were implanted into SCID mice and deeply invaded the cartilage did not show significant expression of PTEN after 60 days. The drop in the percentage of PTEN expressing cells from the original cell cultures to the SCID mouse implants is of interest as this observation goes along with data from previous studies that have shown the prominent expression of activation-related molecules in the SCID mice implants that in vivo are found predominantly in the lining layer. Therefore, our data point to endogenous mechanisms rather than to the influence of exogenous human cytokines or factors in the downregulation of PTEN. Low expression of PTEN may belong to the features that distinguish between the activated phenotype of RA-SF and the sublining, proliferating but nondestructive cells.
PMCID: PMC17804  PMID: 11219390
rheumatoid arthritis; synovial membrane; fibroblasts; PTEN tumour suppressor; severe combined immunodeficiency (SCID) mouse model; cartilage destruction; in situ hybridization
7.  Cytokine, activation marker, and chemokine receptor expression by individual CD4+ memory T cells in rheumatoid arthritis synovium 
Arthritis Research  2000;2(5):415-423.
IL-10, IL-13, IFN-γ, tumor necrosis factor (TNF)-α, LT-α, CD154, and TNF-related activation-induced cytokine (TRANCE) were expressed by 2-20% of rheumatoid arthritis (RA) synovial tissue CD4+ memory T cells, whereas CD4+ cells that produced IL-2, IL-4, or IL-6 were not detected. Expression of none of these molecules by individual CD4+ cells correlated with the exception of TRANCE and IL-10, and TRANCE and TNF-α . A correlation between expression of IL-10 and CCR7, LT-α and CCR6, IFN-γ and CCR5, and TRANCE and CXCR4 was also detected.
Introduction:
In RA large numbers of CD4+ memory T cells infiltrate the inflamed synovium [1,2,3]. The accumulated CD4+ memory T cells in the RA synovium appear to be activated, because they express cytokines and activation markers [4,5,6,7,8]. Expressed cytokines and activation markers should play important roles in the pathogenesis of RA. However, the frequency of cytokine expression by RA synovial CD4+ T cells has not been analyzed accurately. Recently, the roles of chemokine and chemokine receptor interactions in T-cell migration have been intensively examined. Interactions of chemokine and chemokine receptors might therefore be important in the accumulation of the CD4+ T cells in the RA synovium. Accordingly, correlation of cytokine and chemokine receptor expression might be important in delineating the function and potential means of accumulation of individual CD4+ memory T cells in the RA synovium.
In the present study we analyzed cytokine (IL-2, IL-4, IL-6, IL-10, IL-13, IFN-γ , TNF-α , and LT-α ), activation marker (CD154 [CD40 ligand] and TRANCE - also called receptor activator of nuclear factor κ B ligand [RANKL] or osteoclast differentiation factor [ODF]), and chemokine receptor expression by individual CD4+ memory T cells isolated from rheumatoid synovium and blood. To achieve this we employed a single-cell reverse transcription (RT) polymerase chain reaction (PCR) technique. This technique made it possible to correlate mRNAs expressed by individual CD4+ memory T cells in the synovium and blood.
Materials and method:
Synovial tissues from three RA patients and peripheral blood mononuclear cells from two RA patients and a normal donor were analyzed.
Cytokine (IL-2, IL-4, IL-6, IL-10, IL-13, IFN-γ, TNF-α, and LT-α ) and activation marker (CD154 and TRANCE) expression by individual CD4+CD45RO+ T cells from RA synovium or blood were analyzed using a single-cell RT-PCR. In brief, single CD4+CD45RO+T cells was sorted into each well of a 96-well PCR plate using a flow cytometer. cDNA from individual cells was prepared, and then the cDNA was nonspecifically amplified. The product was then amplified by PCR using gene-specific primers to analyze cytokine and activation marker expression.
Results:
Cytokine and activation marker expression by individual CD4+CD45RO+T cells from RA synovial tissues was analyzed using a single-cell RT-PCR method. Expression of mRNAs was analyzed in 152 individual synovial tissue CD4+CD45RO+ T cells sorted from three RA patients in which T-cell receptor (TCR) Cβ mRNA was detected. Frequencies of CD4+ memory T cells expressing cytokine and activation marker mRNA in RA synovium are shown in Table 1. IL-2, IL-4, and IL-6 were not expressed by the synovial tissue CD4+CD45RO+ T cells, whereas 2-20% of cells expressed the other cytokine mRNAs.
Few correlations between cytokine and activation marker mRNAs were observed. Notably, no cells contained both IFN-γ and LT-α mRNAs, cytokines that are thought to define the T-helper (Th)1 phenotype [9]. However, the frequency of TRANCE-positive cells in IL-10-positive cells was significantly higher than that in IL-10-negative cells (Table 2). Moreover, the frequency of TRANCE-positive cells in TNF-α-positive cells was also significantly higher than that in TNF-α-negative cells.
Varying percentages of CD4+ memory T cells expressed CC and CXC chemokine receptors. The frequency of CCR5-positive cells in IFN-γ-positive cells was significantly higher than that in IFN-γ-negative cells, whereas the frequency of CCR6-positive cells in LT-α-positive cells was significantly higher than that in LT-α-negative cells, and the frequency of CCR7-positive cells in IL-10-positive cells was significantly higher than that in IL-10-negative cells. Furthermore, the frequency of CXCR4-positive cells in TRANCE-positive cells was significantly higher than that in TRANCE-negative cells.
Expression of cytokine and activation marker mRNAs was also analyzed in 48 individual peripheral blood CD4+CD45RO+ T cells from two RA patients. IL-2, IL-4, IL-6, and LT-α were not expressed by the peripheral CD4+CD45RO+ T cells, whereas 4-17% of cells expressed the other markers. The most striking difference between synovial tissue and peripheral blood CD4+ memory T cells was the presence of LT-α expression in the former, but not in the latter. IFN-γ and TNF-α were not expressed by normal peripheral blood CD4+ memory T cells, although they were expressed by RA peripheral blood CD4+ memory T cells.
Discussion:
The present study employed a single-cell PCR technology to analyze cytokine expression by unstimulated RA synovial tissue CD4+ memory T cells immediately after isolation, without in vitro manipulation. The results confirm the Th1 nature of rheumatoid inflammation. It is noteworthy that no individual synovial CD4+ memory T cells expressed both IFN-γ and LT-α mRNAs, even though these are the prototypic Th1 cytokines [9]. These results imply that, in the synovium, regulation of IFN-γ and LT-α must vary in individual cells, even though both Th1 cytokines can be produced.
The present data showed that CCR5 expression correlated with IFN-γ but not with LT-α expression by synovial CD4+ memory T cells. It has been reported that CCR5 expression is upregulated in RA synovial fluid and synovial tissue T cells [10,11,12] and that CCR5 Δ 32 deletion may have an influence on clinical manifestations of RA [13], suggesting that CCR5 might play an important role in RA. Recently, it has been claimed that CCR5 was preferentially expressed by Th1 cell lines [14,15]. However, in the present study CCR5 was not expressed by all IFN-γ-expressing cells. Moreover, CCR5 expression did not correlate with expression of LT-α by RA synovial CD4+ memory T cells. Therefore, it is unclear whether CCR5 is a marker of Th1 cells in RA synovium.
IL-10 expression correlated with CCR7 expression by RA synovial CD4+ memory T cells. Recently, it was reported [16] that in the blood CCR7+CD4+ memory T cells express lymph-node homing receptors and lack immediate effector function, but efficiently stimulate dendritic cells. These cells may play a unique role in the synovium as opposed to in the blood. By producing IL-10, they might have an immunoregulatory function. In addition, IL-10 expression also correlated with expression of TRANCE. Although it is possible that IL-10 produced by these cells inhibited T-cell activation in the synovium, TRANCE expressed by these same cells might function to activate dendritic cells and indirectly stimulate T cells, mediating inflammation in the synovium. These results imply that individual T cells in the synovium might have different, and sometimes opposite functional activities.
LT-α expression correlated with CCR6 expression by synovial CD4+ memory T cells. It has been reported that CCR6 is expressed by resting peripheral memory T cells [17], whereas LT-α expression is associated with the presence of lymphocytic aggregates in synovial tissue [7]. The correlation between the expression of these two markers therefore suggests the possibility that CCR6 may play a role in the development of aggregates of CD4+ T cells that are characteristically found in rheumatoid synovium.
TRANCE is known to be expressed by activated T cells, and can stimulate dendritic cells and osteoclasts [18]. Of note, TRANCE-mediated activation of osteoclasts has recently been shown [19] to play an important role in the damage to bone that is found in experimental models of inflammatory arthritis. It is therefore of interest that TRANCE was expressed by 3-16% of the RA synovial CD4+ memory T cells. Of note, 67% of TNF-α-positive cells expressed TRANCE. In concert, TNF-α and TRANCE expressed by this subset of CD4+ memory T cells might make them particularly important in mediating the bony erosions that are characteristic of RA.
Interestingly, there was a correlation between expression of IFN-γ and IL-10 in RA peripheral blood CD4+ memory T cells. In RA peripheral blood, CD154 expression correlated with that of CXCR3 by CD4+ memory T cells. It has been claimed [15] that CXCR3 is preferentially expressed by in vitro generated Th1 cells. However, in the present study CXCR3 did not correlate with IFN-γ expression. Although IFN-γ and TNF-α mRNAs were expressed in vivo by peripheral blood CD4+ T cells from RA patients, LT-α mRNA was not detected, whereas IFN-γ , TNF-α , and LT-α were not detected in samples from healthy donors. These findings indicate that RA peripheral blood CD4+ memory T cells are stimulated in vivo, although they do not express LT-α mRNA. The present studies indicate that the frequencies of CD4+ memory T cells that expressed IFN-γ in the blood and in the synovium are comparable. These results imply that activated CD4+ memory T cells migrate between blood and synovium, although the direction of the trafficking is unknown. The presence of LT-α mRNA in synovium, but not in blood, indicates that CD4+ memory cells are further activated in the synovium, and that these activated CD4+ memory T cells are retained in the synovium until LT-α mRNA decreases.
In conclusion, CD4+ memory T cells are biased toward Th1 cells in RA synovium and peripheral blood. In the synovium, IFN-γ and LT-α were produced by individual cells, whereas in the rheumatoid blood no LT-α-producing cells were detected. Furthermore, there were modest correlations between individual cells that expressed particular cytokines, such as IL-10, and certain chemokine receptor mRNAs.
PMCID: PMC17818  PMID: 11056676
chemokine receptor; cytokine; rheumatoid arthritis; T lymphocyte
8.  Production of angiotensin converting enzyme by rheumatoid synovial membrane. 
Annals of the Rheumatic Diseases  1992;51(4):476-480.
Vascular proliferation and mononuclear cell infiltration are prominent changes observed in synovium from actively inflamed joints of patients with rheumatoid arthritis. Angiotensin converting enzyme (ACE) is a halide activated peptidase produced mainly by endothelial cells and by activated monocytes. It has been proposed that levels of ACE activity in synovial fluid might reflect changes in membrane vascularity, the degree of monocyte infiltration, or the thickness of the lining layer. In this study, ACE activity in serum and synovial fluid samples from 18 patients with inflammatory arthritis was measured and compared with levels in 12 control subjects with non-inflammatory arthritis. Although serum levels were similar in the two groups, ACE activity in synovial fluid was significantly increased in the group with inflammatory arthritis compared with controls (mean (SE) 37 (5) v 19 (3)). Staining of synovial membranes from patients with rheumatoid arthritis with a monoclonal antibody to ACE localised ACE to the endothelium and to mononuclear cells of macrophage origin. ACE activity was then measured in supernatants of synovial membrane from patients with rheumatoid arthritis after one and seven days of culture. A significant increase in ACE activity was observed after seven days of culture (mean (SE) day 1, 17 (5) v day 7, 25 (3)). Levels of ACE activity, however, did not correlate with the lining layer thickness, with the number of macrophages per square millimetre, nor with the number of blood vessels per square millimetre of synovial tissue. No correlation was observed either between levels of ACE in the supernatant of synovial membrane and levels of interleukin 1 or interleukin 6. In conclusion, ACE is produced by the synovial membrane of patients with rheumatoid arthritis and is localised to monocytes and endothelial cells. Levels of activity do not directly reflect membrane vascularity, monocyte or macrophage number, or the thickness of the lining layer.
Images
PMCID: PMC1004695  PMID: 1316742
9.  Active synovial matrix metalloproteinase-2 is associated with radiographic erosions in patients with early synovitis 
Arthritis Research  2000;2(2):145-153.
Serum and synovial tissue expression of the matrix metalloproteinase (MMP)-2 and -9 and their molecular regulators, MMP-14 and TIMP-2 was examined in 28 patients with inflammatory early synovitis and 4 healthy volunteers and correlated with the presence of erosions in the patients. Immunohistological staining of MMP-2, MMP-14 and TIMP-2 localized to corresponding areas in the synovial lining layer and was almost absent in normal synovium. Patients with radiographic erosions had significantly higher levels of active MMP-2 than patients with no erosions, suggesting that activated MMP-2 levels in synovial tissue may be a marker for a more aggressive synovial lesion.
Introduction:
In cancer the gelatinases [matrix metalloproteinase (MMP)-2 and MMP-9] have been shown to be associated with tissue invasion and metastatic disease. In patients with inflammatory arthritis the gelatinases are expressed in the synovial membrane, and have been implicated in synovial tissue invasion into adjacent cartilage and bone. It is hypothesized that an imbalance between the activators and inhibitors of the gelatinases results in higher levels of activity, enhanced local proteolysis, and bone erosion.
Objectives:
To determine whether the expression and activity levels of MMP-2 and MMP-9, and their regulators MMP-14 and tissue inhibitor of metalloproteinase (TIMP), are associated with early erosion formation in patients with synovitis of recent onset.
Patients and method:
A subset of 66 patients was selected from a larger early synovitis cohort on the basis of tissue availability for the study of synovial tissue and serum gelatinase expression. Patients with peripheral joint synovitis of less than 1 years' duration were evaluated clinically and serologically on four visits over a period of 12 months. At the initial visit, patients underwent a synovial tissue biopsy of one swollen joint, and patients had radiographic evaluation of hands and feet initially and at 1year. Serum MMP-1, MMP-2, MMP-9, MMP-14, and TIMP-1 and TIMP-2 levels were determined, and synovial tissue was examined by immunohistology for the expression of MMP-2 and MMP-9, and their molecular regulators. Gelatinolytic activity for MMP-2 and MMP-9 was quantified using a sensitive, tissue-based gel zymography technique. Four healthy individuals underwent closed synovial biopsy and their synovial tissues were similarly analyzed.
Results:
Of the 66 patients studied, 45 fulfilled American College of Rheumatology criteria for rheumatoid arthritis (RA), with 32 (71%) being rheumatoid factor positive. Of the 21 non-RA patients, seven had a spondylarthropathy and 14 had undifferentiated arthritis. Radiographically, 12 of the RA patients had erosions at multiple sites by 1 year, whereas none of the non-RA patients had developed erosive disease of this extent. In the tissue, latent MMP-2 was widely expressed in the synovial lining layer and in areas of stromal proliferation in the sublining layer and stroma, whereas MMP-9 was expressed more sparsely and focally. MMP-14, TIMP-2, and MMP-2 were all detected in similar areas of the lining layer on consecutive histologic sections. Tissue expression of MMP-14, the activator for pro-MMP-2, was significantly higher in RA than in non-RA patients (8.4 ± 5 versus 3.7 ± 4 cells/high-power field; P = 0.009). In contrast, the expression of TIMP-2, an inhibitor of MMP-2, was lower in the RA than in the non-RA samples (25 ± 12 versus 39 ± 9 cells/high-power field; P = 0.01). Synovial tissue expressions of MMP-2, MMP-14, and TIMP-2 were virtually undetectable in normal synovial tissue samples. The synovial tissue samples of patients with erosive disease had significantly higher levels of active MMP-2 than did those of patients without erosions (Fig. 1). Tissue expression of MMP-2 and MMP-9, however, did not correlate with the serum levels of these enzymes.
With the exception of serum MMP-2, which was not elevated over normal, serum levels of all of the other MMPs and TIMPs were elevated to varying degrees, and were not predictive of erosive disease. Interestingly, MMP-1 and C-reactive protein, both of which were associated with the presence of erosions, were positively correlated with each other (r = 0.42; P < 0.001).
Discussion:
MMP-2 and MMP-9 are thought to play an important role in the evolution of joint erosions in patients with an inflammatory arthritis. Most studies have concentrated on the contribution of MMP-9 to the synovitis, because synovial fluid and serum MMP-9 levels are markedly increased in inflammatory arthropathies. Previously reported serum levels of MMP-9 have varied widely. In the present sample of patients with synovitis of recent onset, serum MMP-9 levels were elevated in only 21%. Moreover, these elevations were not specific for RA, the tissue expression of MMP-9 was focal, and the levels of MMP-9 activity were not well correlated with early erosions. Although serum MMP-2 levels were not of prognostic value, high synovial tissue levels of MMP-2 activity were significantly correlated with the presence of early erosions. This may reflect augmented activation of MMP-2 by the relatively high levels of MMP-14 and low levels of TIMP-2 seen in these tissues. We were able to localize the components of this trimolecular complex to the synovial lining layer in consecutive tissue sections, a finding that is consistent with their colocalization.
In conclusion, we have provided evidence that active MMP-2 complexes are detectable in the inflamed RA synovium and may be involved in the development of early bony erosions. These results suggest that strategies to inhibit the activation of MMP-2 may have the potential for retarding or preventing early erosions in patients with inflammatory arthritis.
PMCID: PMC17808  PMID: 11062605
early synovitis; erosion; metalloproteinase; matrix metalloproteinase-2; rheumatoid arthritis
10.  In vitro model for the analysis of synovial fibroblast-mediated degradation of intact cartilage 
Introduction
Activated synovial fibroblasts are thought to play a major role in the destruction of cartilage in chronic, inflammatory rheumatoid arthritis (RA). However, profound insight into the pathogenic mechanisms and the impact of synovial fibroblasts in the initial early stages of cartilage destruction is limited. Hence, the present study sought to establish a standardised in vitro model for early cartilage destruction with native, intact cartilage in order to analyse the matrix-degrading capacity of synovial fibroblasts and their influence on cartilage metabolism.
Methods
A standardised model was established by co-culturing bovine cartilage discs with early-passage human synovial fibroblasts for 14 days under continuous stimulation with TNF-α, IL-1β or a combination of TNF-α/IL-1β. To assess cartilage destruction, the co-cultures were analysed by histology, immunohistochemistry, electron microscopy and laser scanning microscopy. In addition, content and/or neosynthesis of the matrix molecules cartilage oligomeric matrix protein (COMP) and collagen II was quantified. Finally, gene and protein expression of matrix-degrading enzymes and pro-inflammatory cytokines were profiled in both synovial fibroblasts and cartilage.
Results
Histological and immunohistological analyses revealed that non-stimulated synovial fibroblasts are capable of demasking/degrading cartilage matrix components (proteoglycans, COMP, collagen) and stimulated synovial fibroblasts clearly augment chondrocyte-mediated, cytokine-induced cartilage destruction. Cytokine stimulation led to an upregulation of tissue-degrading enzymes (aggrecanases I/II, matrix-metalloproteinase (MMP) 1, MMP-3) and pro-inflammatory cytokines (IL-6 and IL-8) in both cartilage and synovial fibroblasts. In general, the activity of tissue-degrading enzymes was consistently higher in co-cultures with synovial fibroblasts than in cartilage monocultures. In addition, stimulated synovial fibroblasts suppressed the synthesis of collagen type II mRNA in cartilage.
Conclusions
The results demonstrate for the first time the capacity of synovial fibroblasts to degrade intact cartilage matrix by disturbing the homeostasis of cartilage via the production of catabolic enzymes/pro-inflammatory cytokines and suppression of anabolic matrix synthesis (i.e., collagen type II). This new in vitro model may closely reflect the complex process of early stage in vivo destruction in RA and help to elucidate the role of synovial fibroblasts and other synovial cells in this process, and the molecular mechanisms involved in cartilage degradation.
doi:10.1186/ar2618
PMCID: PMC2688258  PMID: 19226472
11.  The role of synovial macrophages and macrophage-produced cytokines in driving aggrecanases, matrix metalloproteinases, and other destructive and inflammatory responses in osteoarthritis 
There is an increasing body of evidence that synovitis plays a role in the progression of osteoarthritis and that overproduction of cytokines and growth factors from the inflamed synovium can influence the production of degradative enzymes and the destruction of cartilage. In this study, we investigate the role of synovial macrophages and their main proinflammatory cytokines, interleukin (IL)-1 and tumour necrosis factor-alpha (TNF-α), in driving osteoarthritis synovitis and influencing the production of other pro- and anti-inflammatory cytokines, production of matrix metalloproteinases, and expression of aggrecanases in the osteoarthritis synovium. We established a model of cultures of synovial cells from digested osteoarthritis synovium derived from patients undergoing knee or hip arthroplasties. By means of anti-CD14-conjugated magnetic beads, specific depletion of osteoarthritis synovial macrophages from these cultures could be achieved. The CD14+-depleted cultures no longer produced significant amounts of macrophage-derived cytokines like IL-1 and TNF-α. Interestingly, there was also significant downregulation of several cytokines, such as IL-6 and IL-8 (p < 0.001) and matrix metalloproteinases 1 and 3 (p < 0.01), produced chiefly by synovial fibroblasts. To investigate the mechanisms involved, we went on to use specific downregulation of IL-1 and/or TNF-α in these osteoarthritis cultures of synovial cells. The results indicated that neutralisation of both IL-1 and TNF-α was needed to achieve a degree of cytokine (IL-6, IL-8, and monocyte chemoattractant protein-1) and matrix metalloproteinase (1, 3, 9, and 13) inhibition, as assessed by enzyme-linked immunosorbent assay and by reverse transcription-polymerase chain reaction (RT-PCR), similar to that observed in CD14+-depleted cultures. Another interesting observation was that in these osteoarthritis cultures of synovial cells, IL-1β production was independent of TNF-α, in contrast to the situation in rheumatoid arthritis. Using RT-PCR, we also demonstrated that whereas the ADAMTS4 (a disintegrin and metalloprotease with thrombospondin motifs 4) aggrecanase was driven mainly by TNF-α, ADAMTS5 was not affected by neutralisation of IL-1 and/or TNF-α. These results suggest that, in the osteoarthritis synovium, both inflammatory and destructive responses are dependent largely on macrophages and that these effects are cytokine-driven through a combination of IL-1 and TNF-α.
doi:10.1186/ar2099
PMCID: PMC1794533  PMID: 17177994
12.  The effects of 1α,25-dihydroxyvitamin D3 on matrix metalloproteinase and prostaglandin E2 production by cells of the rheumatoid lesion 
Arthritis Research  1999;1(1):63-70.
The biologically active metabolite of vitamin D3, 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], acts through vitamin D receptors, which were found in rheumatoid tissues in the present study. IL-1β-activated rheumatoid synovial fibroblasts and human articular chondrocytes were shown to respond differently to exposure to 1α,25(OH)2D3, which has different effects on the regulatory pathways of specific matrix metalloproteinases and prostaglandin E2.
Introduction:
1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], the biologically active metabolite of vitamin D3, acts through an intracellular vitamin D receptor (VDR) and has several immunostimulatory effects. Animal studies have shown that production of some matrix metalloproteinases (MMPs) may be upregulated in rat chondrocytes by administration of 1α,25(OH)2D3; and cell cultures have suggested that 1α,25(OH)2D3 may affect chondrocytic function. Discoordinate regulation by vitamin D of MMP-1 and MMP-9 in human mononuclear phagocytes has also been reported. These data suggest that vitamin D may regulate MMP expression in tissues where VDRs are expressed. Production of 1α,25(OH)2D3 within synovial fluids of arthritic joints has been shown and VDRs have been found in rheumatoid synovial tissues and at sites of cartilage erosion. The physiological function of 1α,25(OH)2D3 at these sites remains obscure. MMPs play a major role in cartilage breakdown in the rheumatoid joint and are produced locally by several cell types under strict control by regulatory factors. As 1α,25(OH)2D3 modulates the production of specific MMPs and is produced within the rheumatoid joint, the present study investigates its effects on MMP and prostaglandin E2 (PGE2) production in two cell types known to express chondrolytic enzymes.
Aims:
To investigate VDR expression in rheumatoid tissues and to examine the effects of 1α,25-dihydroxyvitamin D3 on cultured rheumatoid synovial fibroblasts (RSFs) and human articular chondrocytes (HACs) with respect to MMP and PGE2 production.
Methods:
Rheumatoid synovial tissues were obtained from arthroplasty procedures on patients with late-stage rheumatoid arthritis; normal articular cartilage was obtained from lower limb amputations. Samples were embedded in paraffin, and examined for presence of VDRs by immunolocalisation using a biotinylated antibody and alkaline-phosphatase-conjugated avidin-biotin complex system. Cultured synovial fibroblasts and chondrocytes were treated with either 1α,25(OH)2D3, or interleukin (IL)-1β or both. Conditioned medium was assayed for MMP and PGE2 by enzyme-linked immunosorbent assay (ELISA), and the results were normalised relative to control values.
Results:
The rheumatoid synovial tissue specimens (n = 18) immunostained for VDRs showed positive staining but at variable distributions and in no observable pattern. VDR-positive cells were also observed in association with some cartilage-pannus junctions (the rheumatoid lesion). MMP production by RSFs in monolayer culture was not affected by treatment with 1α,25(OH)2D3 alone, but when added simultaneously with IL-1β the stimulation by IL-1β was reduced from expected levels by up to 50%. In contrast, 1α,25(OH)2D3 had a slight stimulatory effect on basal production of MMPs 1 and 3 by monolayer cultures of HACs, but stimulation of MMP-1 by IL-1β was not affected by the simultaneous addition of 1α,25(OH)2D3 whilst MMP-3 production was enhanced (Table 1). The production of PGE2 by RSFs was unaffected by 1α,25(OH)2D3 addition, but when added concomitantly with IL-1β the expected IL-1 β-stimulated increase was reduced to almost basal levels. In contrast, IL-1β stimulation of PGE2 in HACs was not affected by the simultaneous addition of 1α,25(OH)2D3 (Table 2). Pretreatment of RSFs with 1α,25(OH)2D3 for 1 h made no significant difference to IL-1β-induced stimulation of PGE2, but incubation for 16 h suppressed the expected increase in PGE2 to control values. This effect was also noted when 1α,25(OH)2D3 was removed after the 16h and the IL-1 added alone. Thus it appears that 1α,25(OH)2D3 does not interfere with the IL-1β receptor, but reduces the capacity of RSFs to elaborate PGE2 after IL-1β induction.
Discussion:
Cells within the rheumatoid lesion which expressed VDR were fibroblasts, macrophages, lymphocytes and endothelial cells. These cells are thought to be involved in the degradative processes associated with rheumatoid arthritis (RA), thus providing evidence of a functional role of 1α,25(OH)2D3 in RA. MMPs may play important roles in the chondrolytic processes of the rheumatoid lesion and are known to be produced by both fibroblasts and chondrocytes. The 1α,25(OH)2D3 had little effect on basal MMP production by RSFs, although more pronounced differences were noted when IL-1β-stimulated cells were treated with 1α,25(OH)2D3, with the RSF and HAC showing quite disparate responses. These opposite effects may be relevant to the processes of joint destruction, especially cartilage loss, as the ability of 1α,25(OH)2D3 to potentiate MMP-1 and MMP-3 expression by 'activated' chondrocytes might facilitate intrinsic cartilage chondrolysis in vivo. By contrast, the MMP-suppressive effects observed for 1α,25(OH)2D3 treatment of 'activated' synovial fibroblasts might reduce extrinsic chondrolysis and also matrix degradation within the synovial tissue. Prostaglandins have a role in the immune response and inflammatory processes associated with RA. The 1α,25(OH)2D3 had little effect on basal PGE2 production by RSF, but the enhanced PGE2 production observed following IL-1β stimulation of these cells was markedly suppressed by the concomitant addition of 1α,25(OH)2D3. As with MMP production, there are disparate effects of 1α,25(OH)2D3 on IL-1β stimulated PGE2 production by the two cell types; 1α,25(OH)2D3 added concomitantly with IL-1β had no effect on PGE2 production by HACs. In summary, the presence of VDRs in the rheumatoid lesion demonstrates that 1α,25(OH)2D3 may have a functional role in the joint disease process. 1α,25(OH)2D3 does not appear to directly affect MMP or PGE2 production but does modulate cytokine-induced production.
Comparative effects of 1 α,25-dihydroxyvitamin D3 (1 α,25D3) on interleukin (IL)-1-stimulated matrix metalloproteinase (MMP)-1 and MMP-3 production by rheumatoid synovial fibroblasts and human articular chondrocytes in vivo
Data given are normalized relative to control values and are expressed ± SEM for three cultures of each cell type.
Comparative effects of 1α,25-dihydroxyvitamin D3 (1α,25D3) on Interleukin (IL)-1-stimulated prostaglandin E2 production by rheumatoid synovial fibroblasts and human articular chondrocyte in vivo
Data given are normalized relative to control values and are expressed ± SEM for three cultures of each cell type.
PMCID: PMC17774  PMID: 11056661
1α,25-dihydroxyvitamin D3; matrix metalloproteinase; prostaglandin E2; rheumatoid arthritis
13.  The immunoreceptor tyrosine-based activation motif (ITAM) -related factors are increased in synovial tissue and vasculature of rheumatoid arthritic joints 
Arthritis Research & Therapy  2012;14(6):R245.
Introduction
The immunoreceptor tyrosine-based activation motif (ITAM) pathway provides osteoclast co-stimulatory signals and regulates proliferation, survival and differentiation of effector immune cells. In the osteoclast, the receptors Triggering Receptor Expressed on Myeloid cells 2 (TREM2) and Osteoclast Associated Receptor (OSCAR) and their respective adaptor proteins, DAP12 and FcRγ mediate ITAM signals and induce calcium signaling and the crucial transcription factor, NFATc1. In rheumatoid arthritis (RA), OSCAR expression by monocytes is inversely correlated with disease activity. Additionally, serum levels of OSCAR are reduced in RA patients versus healthy controls suggesting that expression and secretion or cleavage of soluble (s) OSCAR is immune modulated. Recent data suggest that endothelial cells may also be a source of OSCAR.
Methods
ITAM receptors, their adaptor proteins, and NFATc1 and cathepsin K were detected in human synovial tissues by immunohistochemistry. Synovial tissues from patients with active RA were compared with tissue from patients in remission, osteoarthritis (OA) patients and healthy individuals. OSCAR was measured by immunoassay in synovial fluids recovered from active RA and OA patients. Endothelial cells were cultured with or without 5 ng/mL TNF-α or IL-1β over 72 hours. Temporal expression of OSCAR mRNA was assessed by qRT PCR and OSCAR protein in the supernatant was measured by ELISA.
Results
Significantly higher (P < 0.05) NFATc1-positive inflammatory cell aggregates were found in active RA tissues than in healthy synovial tissue. Similarly, the percentage of OSCAR, FcRγ, DAP12 and TREM2 positive cells was significantly higher in active RA tissues compared to the healthy synovial tissue. Notably, OSCAR was strongly expressed in the microvasculature of the active RA tissues (9/9), inactive RA (8/9) weakly in OA (4/9) but only in the lumen of healthy synovial tissue (0/8). OSCAR levels were detected in synovial fluids from both RA (47 to 152 ng/mL) and OA (112 to 145 ng/mL) patients. Moreover, OSCAR mRNA expression and soluble OSCAR release was stimulated by TNF-α and IL1-β in cultured endothelial cells.
Conclusions
Increased levels of ITAM related factors were present in synovial tissue from active RA joints compared to OA and healthy joints. OSCAR was strongly expressed by the vasculature of active RA patients and membrane bound and soluble OSCAR was stimulated by inflammatory mediators in endothelial cells in vitro.
doi:10.1186/ar4088
PMCID: PMC3674611  PMID: 23146195
14.  FcgammaR expression on macrophages is related to severity and chronicity of synovial inflammation and cartilage destruction during experimental immune-complex-mediated arthritis (ICA) 
Arthritis Research  2000;2(6):489-503.
We investigated the role of Fcγ receptors (FcγRs) on synovial macrophages in immune-complex-mediated arthritis (ICA). ICA elicited in knee joints of C57BL/6 mice caused a short-lasting, florid inflammation and reversible loss of proteoglycans (PGs), moderate chondrocyte death, and minor erosion of the cartilage. In contrast, when ICA was induced in knee joints of Fc receptor (FcR) γ-chain-/- C57BL/6 mice, which lack functional FcγRI and RIII, inflammation and cartilage destruction were prevented. When ICA was elicited in DBA/1 mice, a very severe, chronic inflammation was observed, and significantly more chondrocyte death and cartilage erosion than in arthritic C57BL/6 mice. The synovial lining and peritoneal macrophages of naïve DBA/1 mice expressed a significantly higher level of FcγRs than was seen in C57BL/6 mice. Moreover, elevated and prolonged expression of IL-1 was found after stimulation of these cells with immune complexes. Zymosan or streptococcal cell walls caused comparable inflammation and only mild cartilage destruction in all strains. We conclude that FcγR expression on synovial macrophages may be related to the severity of synovial inflammation and cartilage destruction during ICA.
Introduction:
Fcγ receptors (FcγRs) present on cells of the haematopoietic lineage communicate with IgG-containing immune complexes that are abundant in the synovial tissue of patients with rheumatoid arthritis (RA). In mice, three classes of FcγR (RI, RII, and RIII) have been described. Binding of these receptors leads to either activation (FcγRI and RIII) or deactivation (FcγRII) of intracellular transduction pathways. Together, the expression of activating and inhibitory receptors is thought to drive immune-complex-mediated diseases.
Earlier studies in our laboratory showed that macrophages of the synovial lining are of utmost importance in the onset and propagation of immune-complex-driven arthritic diseases. Selective depletion of macrophages in the joint downregulated both inflammation and cartilage destruction. As all three classes of FcγR are expressed on synovial macrophages, these cells are among the first that come in contact with immune complexes deposited in the joint. Recently, we observed that when immune complexes were injected into the knee joints of mice, strains susceptible to collagen-type-II arthritis (DBA/1, B10.RIII) developed more severe arthritis than nonsusceptible strains did, or even developed chronic arthritis. One reason why these strains are more susceptible might be their higher levels of FcγRs on macrophage membranes. To test this hypothesis, we investigated the role of FcγRs in inflammation and cartilage damage during immune-complex-mediated arthritis (ICA). First, we studied arthritis and subsequent cartilage damage in mice lacking functional FcγRI and RIII (FcR γ-chain-/- mice). Next, DBA/1 mice, which are prone to develop collagen-type-II arthritis (`collagen-induced arthritis'; CIA) and are hypersensitive to immune complexes, were compared with control C57BL/6 mice as regards cartilage damage and the expression and function of FcγRs on their macrophages.
Aims:
To examine whether FcγR expression on macrophages is related to severity of synovial inflammation and cartilage destruction during immune-complex-mediated joint inflammation.
Methods:
ICA was induced in three strains of mice (FcR γ-chain-/-, C57BL/6, and DBA/1, which have, respectively, no functional FcγRI and RIII, intermediate basal expression of FcγRs, and high basal expression of FcγRs) by passive immunisation using rabbit anti-lysozyme antibodies, followed by poly-L-lysine lysozyme injection into the right knee joint 1 day later. In other experiments, streptococcal-cell-wall (SCW)- or zymosan-induced arthritis was induced by injecting SCW (25 μg) or zymosan (180 μg) directly into the knee joint. At several time points after arthritis induction, knee joints were dissected and studied either histologically (using haematoxylin/eosin or safranin O staining) or immuno-histochemically. The arthritis severity and the cartilage damage were scored separately on an arbitrary scale of 0-3.
FcγRs were immunohistochemically detected using the monoclonal antibody 2.4G2, which detects both FcγRII and RIII. Deposition of IgG and C3c in the arthritic joint tissue was also detected immunohistochemically. Expression of FcγRs by murine peritoneal macrophages was measured using a fluorescence-activated cell sorter (FACS).
Peritoneal macrophages were stimulated using heat-aggregated gamma globulins (HAGGs), and production of IL-1 was measured using a bioassay. To assess the levels of IL-1 and its receptor antagonist (IL-1Ra) during arthritis, tissue was dissected and washed in RPMI medium. Washouts were tested for levels of IL-1 and IL-1Ra using radioimmunoassay and enzyme-linked immunosorbent assay. mRNA was isolated from the tissue, and levels of macrophage inflammatory protein (MIP)-2, monocyte chemoattractant protein (MCP)-1, IL-1, and IL-1Ra were determined using semiquantitative reverse-transcription polymerase chain reaction (RT-PCR).
Results:
ICA induced in knee joints of C57BL/6 mice caused a florid inflammation at day 3 after induction. To investigate whether this arthritis was FcγR-mediated, ICA was induced in FcR γ-chain-/- mice, which lack functional FcγRI and RIII. At day3, virtually no inflammatory cells were found in their knee joints. Levels of mRNA of IL-1, IL-1Ra, MCP-1, and MIP-2, which are involved in the onset of this arthritis, were significantly lower in FcR γ-chain-/- mice than in control C57BL/6 mice. Levels of IL-1 protein were also measured. At 6 h after ICA induction, FcR γ-chain-/- mice and control C57BL/6 mice showed similar IL-1 production as measured by protein level. By 24 h after induction, however, IL-1 production in the FcR γ-chain-/- mice was below the detection limit, whereas the controls were still producing a significant amount. To investigate whether the difference in reaction to immune complexes between the DBA/1 and C57BL/6 mice might be due to variable expression of FcγRs in the knee joint, expression in situ of FcγRs in naïve knee joints of these mice was determined. The monoclonal antibody 2.4G2, which detects both FcγRII and RIII, stained macrophages from the synovial lining of DBA/1 mice more intensely than those from C57BL/6 mice. This finding suggests a higher constitutive expression of FcγRs by macrophages of the autoimmune-prone DBA/1 mice. To quantify the difference in FcγR expression on macrophages of the two strains, we determined the occurrence of FcγRs on peritoneal macrophages by FACS analysis. The levels of FcγR expressed by macrophages were twice as high in the DBA/1 mice as in the C57BL/6 mice (mean fluorescence, respectively, 440 ± 50 and 240 ± 30 intensity per cell). When peritoneal macrophages of both strains were stimulated with immune complexes (HAGGs), we found that the difference in basal FcγR expression was functional. The stimulated macrophages from DBA/1 mice had significantly higher IL-1α levels (120 and 135 pg/ml at 24 and 48 h, respectively) than cells from C57BL/6 mice (45 and 50 pg/ml, respectively).
When arthritis was induced using other arthritogenic triggers than immune complexes (zymosan, SCW), all the mouse strains tested (DBA/1, FcR γ-chain-/-, and C57BL/6) showed similar inflammation, indicating that the differences described above are found only when immune complexes are used to elicit arthritis.
We next compared articular cartilage damage in arthritic joints of the three mouse strains FcR γ-chain-/-, C57BL/6 (intermediate basal expression of FcγRs), and DBA/1 (high basal expression of FcγRs). Three indicators of cartilage damage were investigated: depletion of PGs, chondrocyte death, and erosion of the cartilage matrix. At day 3 after induction of ICA, there was no PG depletion in FcR γ-chain-/- mice, whereas PG depletion in the matrix of the C57BL/6 mice was marked and that in the arthritic DBA/1 mice was even greater. PG depletion was still massive at days 7 and 14 in the DBA/1 mice, whereas by day 14 the PG content was almost completely restored in knee joints of the C57BL/6 mice. Chondrocyte death and erosion of cartilage matrix, two indicators of more severe cartilage destruction, were significantly higher in the DBA/1 than in the C57BL/6 mice, while both indicators were completely absent in the FcR γ-chain-/- mice. Again, when arthritis was induced using other triggers (SCW, zymosan), all strains showed similar PG depletion and no chondrocyte death or matrix erosion. These findings underline the important role of immune complexes and FcγRs in irreversible cartilage damage.
Discussion:
Our findings indicate that inflammation and subsequent cartilage damage caused by immune complexes may be related to the occurrence of FcγRs on macrophages. The absence of functional FcγRI and RIII prevented inflammation and cartilage destruction after induction of ICA, whereas high basal expression of FcγRs on resident joint macrophages of similarly treated mice susceptible to autoimmune arthritis was correlated with markedly more synovial inflammation and cartilage destruction. The difference in joint inflammation between the three strains was not due to different susceptibilities to inflammation per se, since intra-articular injection of zymosan or SCW caused comparable inflammation. Although extensive inflammatory cell mass was found in the synovium of all strains after intra-articular injection of zymosan, no irreversible cartilage damage (chondrocyte death or matrix erosion) was found. ICA induced in C57BL/6 and DBA/1 mice did cause irreversible cartilage damage at later time points, indicating that immune complexes and FcγRs play an important role in inducing irreversible cartilage damage. Macrophages communicate with immune complexes via Fcγ receptors. Absence of functional activating receptors completely abrogates the synovial inflammation, as was shown after ICA induction in FcR γ-chain-/- mice. However, the γ-chain is essential not only in FcγRI and RIII but also for FcεRI (found on mast cells) and the T cell receptor (TcR)-CD3 (Tcells) complex of γδT cells. However, T, B, or mast cells do not play a role in this arthritis that is induced by passive immunisation. Furthermore, this effect was not caused by a difference in clearance of IgG or complement deposition in the tissue. In this study, DBA/1 mice, which are susceptible to collagen-induced autoimmune arthritis and in a recent study have been shown to react hypersensitively to immune complexes, are shown to express higher levels of FcγRs on both synovial and peritoneal macrophages. Because antibodies directed against the different subclasses of FcγR are not available, no distinction could be made between FcγRII and RIII. Genetic differences in DBA/1 mice in genes coding for or regulating FcγRs may be responsible for altered FcγR expression. If so, these mouse strains would have a heightened risk for immune-complex-mediated diseases.
To provide conclusive evidence for the roles of the various classes of FcγR during ICA, experiments are needed in which FcγRs are blocked with specific antibodies, or in which knockout mice lacking one specific class of FcγR are used. The only available specific antibody to FcγR (2.4G2) has a stimulatory effect on cells once bound to the receptor, and therefore cannot be used in blocking experiments. Experiments using specific knockout mice are now being done in our laboratory.
Macrophages are the dominant type of cell present in chronic inflammation during RA and their number has been shown to correlate well with severe cartilage destruction. Apart from that, in humans, these synovial tissue macrophages express activating FcRs, mainly FcγIIIa, which may lead to activation of these macrophages by IgG-containing immune complexes. The expression of FcRs on the surface of these cells may have important implications for joint inflammation and severe cartilage destruction and therefore FCRs may constitute a new target for therapeutic intervention.
PMCID: PMC17821  PMID: 11056679
autoimmunity; cytokines; Fc receptors; inflammation; macrophages
15.  Specificity of T cells in synovial fluid: high frequencies of CD8+ T cells that are specific for certain viral epitopes 
Arthritis Research  2000;2(2):154-164.
CD8+ T cells dominate the lymphocyte population in synovial fluid in chronic inflammatory arthritis. It is known that these CD8+ T cells are often clonally or oligoclonally expanded, but their specificity and their relevance to the pathogenesis of joint disease has remained unclear. We found that as many as 15.5% of synovial CD8+ T cells may be specific for a single epitope from an Epstein-Barr virus lytic cycle protein. The virus-specific T cells within the joint showed increased expression of markers of activation and differentiation compared with those in the periphery, and retained their functional capacity to secrete proinflammatory cytokines on stimulation. These activated, virus-specific CD8+ T cells could therefore interact with synoviocytes, either by cell-cell contact or by a cytokine network, and play a 'bystander' role in the maintenance of inflammation in patients with arthritis.
Introduction:
Epstein-Barr virus (EBV) is transmitted orally, replicates in the oropharynx and establishes life-long latency in human B lymphocytes. T-cell responses to latent and lytic/replicative cycle proteins are readily detectable in peripheral blood from healthy EBV-seropositive individuals. EBV has also been detected within synovial tissue, and T-cell responses to EBV lytic proteins have been reported in synovial fluid from a patient with rheumatoid arthritis (RA). This raises the question regarding whether T cells specific for certain viruses might be present at high frequencies within synovial fluid and whether such T cells might be activated or able to secrete cytokines. If so, they might play a 'bystander' role in the pathogenesis of inflammatory joint disease.
Objectives:
To quantify and characterize T cells that are specific for epitopes from EBV, cytomegalovirus (CMV) and influenza in peripheral blood and synovial fluid from patients with arthritis.
Methods:
Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were obtained from patients with inflammatory arthritis (including those with RA, osteoarthritis, psoriatic arthritis and reactive arthritis). Samples from human leucocyte antigen (HLA)-A2-positive donors were stained with fluorescent-labelled tetramers of HLA-A2 complexed with the GLCTLVAML peptide epitope from the EBV lytic cycle protein BMLF1, the GILGFVFTL peptide epitope from the influenza A matrix protein, or the NLVPMVATV epitope from the CMV pp65 protein. Samples from HLA-B8-positive donors were stained with fluorescent-labelled tetramers of HLA-B8 complexed with the RAKFKQLL peptide epitope from the EBV lytic protein BZLF1 or the FLRGRAYGL peptide epitope from the EBV latent protein EBNA3A. All samples were costained with an antibody specific for CD8. CD4+ T cells were not analyzed. Selected samples were costained with antibodies specific for cell-surface glycoproteins, in order to determine the phenotype of the T cells within the joint and the periphery. Functional assays to detect release of IFN-γ or tumour necrosis factor (TNF)-α were also performed on some samples.
Results:
The first group of 15 patients included 10 patients with RA, one patient with reactive arthritis, one patient with psoriatic arthritis and three patients with osteoarthritis. Of these, 11 were HLA-A2 positive and five were HLA-B8 positive. We used HLA-peptide tetrameric complexes to analyze the frequency of EBV-specific T cells in PBMCs and SFMCs (Figs 1 and 2). Clear enrichment of CD8+ T cells specific for epitopes from the EBV lytic cycle proteins was seen within synovial fluid from almost all donors studied, including patients with psoriatic arthritis and osteoarthritis and those with RA. In donor RhA6, 9.5% of CD8+ SFMCs were specific for the HLA-A2 restricted GLCTLVAML epitope, compared with 0.5% of CD8+ PBMCs. Likewise in a donor with osteoarthritis (NR4), 15.5% of CD8+ SFMCs were specific for the HLA-B8-restricted RAKFKQLL epitope, compared with 0.4% of CD8+ PBMCs. In contrast, we did not find enrichment of T cells specific for the HLA-B8-restricted FLRGRAYGL epitope (from the latent protein EBNA3A) within SFMCs compared with PBMCs in any donors. In selected individuals we performed ELISpot assays to detect IFN-γ secreted by SFMCs and PBMCs after a short incubation in vitro with peptide epitopes from EBV lytic proteins. These assays confirmed enrichment of T cells specific for epitopes from EBV lytic proteins within synovial fluid and showed that subpopulations of these cells were able to secrete proinflammatory cytokines after short-term stimulation.
We used a HLA-A2/GILGFVFTL tetramer to stain PBMCs and SFMCs from six HLA-A2-positive patients. The proportion of T cells specific for this influenza epitope was low (<0.2%) in all donors studied, and we did not find any enrichment within SFMCs.
We had access to SFMCs only from a second group of four HLA-A2-positive patients with RA. A tetramer of HLA-A2 complexed to the NLVPMVATV epitope from the CMV pp65 protein reacted with subpopulations of CD8+ SFMCs in all four donors, with frequencies of 0.2, 0.5, 2.3 and 13.9%. SFMCs from all four donors secreted TNF after short-term incubation with COS cells transfected with HLA-A2 and pp65 complementary DNA. We analyzed the phenotype of virus-specific cells within PBMCs and SFMCs in three donors. The SFMC virus-specific T cells were more highly activated than those in PBMCs, as evidenced by expression of high levels of CD69 and HLA-DR. A greater proportion of SFMCs were CD38+, CD62L low, CD45RO bright, CD45RA dim, CD57+ and CD28- when compared with PBMCs.
Discussion:
This work shows that T cells specific for certain epitopes from viral proteins are present at very high frequencies (up to 15.5% of CD8+ T cells) within SFMCs taken from patients with inflammatory joint disease. This enrichment does not reflect a generalized enrichment for the 'memory pool' of T cells; we did not find enrichment of T cells specific for the GILGFVFTL epitope from influenza A or for the FLRGRAYGL epitope from the EBV latent protein EBNA3A, whereas we found clear enrichment of T cells specific for the GLCTLVAML epitope from the EBV lytic protein BMLF1 and for the RAKFKQLL epitope from the EBV lytic protein BZLF1.
The enrichment might reflect preferential recruitment of subpopulations of virus-specific T cells, perhaps based on expression of selectins, chemokine receptors or integrins. Alternatively, T cells specific for certain viral epitopes may be stimulated to proliferate within the joint, by viral antigens themselves or by cross-reactive self-antigens. Finally, it is theoretically possible that subpopulations of T cells within the joint are preferentially protected from apoptotic cell death. Whatever the explanation, the virus-specific T cells are present at high frequency, are activated and are able to secrete proinflammatory cytokines. They could potentially interact with synoviocytes and contribute to the maintenance of inflammation within joints in many different forms of inflammatory arthritis.
PMCID: PMC17809  PMID: 11062606
CD8+ T cell; Epstein-Barr virus lytic cycle; human leucocyte antigen peptide tetrameric complex; rheumatoid arthritis; viral immunity
16.  Coexpression and interaction of CXCL10 and CD26 in mesenchymal cells by synergising inflammatory cytokines: CXCL8 and CXCL10 are discriminative markers for autoimmune arthropathies 
Leukocyte infiltration during acute and chronic inflammation is regulated by exogenous and endogenous factors, including cytokines, chemokines and proteases. Stimulation of fibroblasts and human microvascular endothelial cells with the inflammatory cytokines interleukin-1β (IL-1β) or tumour necrosis factor alpha (TNF-α) combined with either interferon-α (IFN-α), IFN-β or IFN-γ resulted in a synergistic induction of the CXC chemokine CXCL10, but not of the neutrophil chemoattractant CXCL8. In contrast, simultaneous stimulation with different IFN types did not result in a synergistic CXCL10 protein induction. Purification of natural CXCL10 from the conditioned medium of fibroblasts led to the isolation of CD26/dipeptidyl peptidase IV-processed CXCL10 missing two NH2-terminal residues. In contrast to intact CXCL10, NH2-terminally truncated CXCL10(3–77) did not induce extracellular signal-regulated kinase 1/2 or Akt/protein kinase B phosphorylation in CXC chemokine receptor 3-transfected cells. Together with the expression of CXCL10, the expression of membrane-bound CD26/dipeptidyl peptidase IV was also upregulated in fibroblasts by IFN-γ, by IFN-γ plus IL-1β or by IFN-γ plus TNF-α. This provides a negative feedback for CXCL10-dependent chemotaxis of activated T cells and natural killer cells. Since TNF-α and IL-1β are implicated in arthritis, synovial concentrations of CXCL8 and CXCL10 were compared in patients suffering from crystal arthritis, ankylosing spondylitis, psoriatic arthritis and rheumatoid arthritis. All three groups of autoimmune arthritis patients (ankylosing spondylitis, psoriatic arthritis and rheumatoid arthritis) had significantly increased synovial CXCL10 levels compared with crystal arthritis patients. In contrast, compared with crystal arthritis, only rheumatoid arthritis patients, and not ankylosing spondylitis or psoriatic arthritis patients, had significantly higher synovial CXCL8 concentrations. Synovial concentrations of the neutrophil chemoattractant CXCL8 may therefore be useful to discriminate between autoimmune arthritis types.
doi:10.1186/ar1997
PMCID: PMC1779382  PMID: 16846531
17.  Borrelia burgdorferi Induces the Production and Release of Proinflammatory Cytokines in Canine Synovial Explant Cultures 
Infection and Immunity  1998;66(1):247-258.
Canine synovial membrane explants were exposed to high- or low-passage Borrelia burgdorferi for 3, 6, 12, and 24 h. Spirochetes received no treatment, were UV light irradiated for 16 h, or were sonicated prior to addition to synovial explant cultures. In explant tissues, mRNA levels for the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-1α (IL-1α), IL-1β, and IL-8 were surveyed semiquantitatively by reverse transcription-PCR. Culture supernatants were examined for numbers of total and motile (i.e., viable) spirochetes, TNF-like and IL-1-like activities, polymorphonuclear neutrophil (PMN) chemotaxis-inducing activities, and IL-8. During exposure to synovial explant tissues, the total number of spirochetes in the supernatants decreased gradually by ∼30%, and the viability also declined. mRNAs for TNF-α, IL-1α, IL-1β, and IL-8 were up-regulated in synovial explant tissues within 3 h after infection with untreated or UV light-irradiated B. burgdorferi, and mRNA levels corresponded to the results obtained with bioassays. During 24 h of coincubation, cultures challenged with untreated or UV light-irradiated spirochetes produced similar levels of TNF-like and IL-1-like activities. In contrast, explant tissues exposed to untreated B. burgdorferi generated significantly higher levels of chemotactic factors after 24 h of incubation than did explant tissues exposed to UV light-treated spirochetes. In identical samples, a specific signal for IL-8 was identified by Western blot analysis. High- and low-passage borreliae did not differ in their abilities to induce proinflammatory cytokines. No difference in cytokine induction between untreated and sonicated high-passage spirochetes was observed, suggesting that fractions of the organism can trigger the production and release of inflammatory mediators. The titration of spirochetes revealed a dose-independent cytokine response, where 103 to 107 B. burgdorferi organisms induced similar TNF-like activities but only 107 spirochetes induced measurable IL-1-like activities. The release of chemotactic factors was dose dependent and was initiated when tissues were infected with at least 105 organisms. We conclude that intact B. burgdorferi or fractions of the bacterium can induce the local up-regulation of TNF-α, IL-1α, and IL-1β in the synovium but that the interaction of viable spirochetes with synovial cells leads to the release of IL-8, which probably is a prime initiator of PMN migration during acute Lyme arthritis.
PMCID: PMC107884  PMID: 9423865
18.  Effects of treatment with a fully human anti-tumour necrosis factor α monoclonal antibody on the local and systemic homeostasis of interleukin 1 and TNFα in patients with rheumatoid arthritis 
Annals of the Rheumatic Diseases  2001;60(7):660-669.
OBJECTIVES—To study the short term effects of a single dose of D2E7, a fully human anti-tumour necrosis factor (TNFα) monoclonal antibody (mAb), on the local and systemic homeostasis of interleukin 1β (IL1β) and TNFα in patients with rheumatoid arthritis (RA).
METHODS—All patients with RA enrolled in a phase I, single dose, placebo controlled study with D2E7 in our centre were studied. Systemic cytokine levels, acute phase reactants, and leucocyte counts were studied at days 0, 1, and 14 after the first administration of anti-TNF mAb (n=39) or placebo (n=11). The cellularity and the expression of IL1 and TNFα in synovial tissue were studied in knee biopsy specimens obtained at baseline and at day 14 in 25 consenting patients.
RESULTS—A single dose of anti-TNF mAb induced a rapid clinical improvement, a decrease in acute phase reaction, and increased lymphocyte counts in patients with active RA. The protein levels of IL1β in the circulation were low and remained unchanged, but the systemic levels of IL1β mRNA (p=0.002) and the concentrations of IL1 receptor antagonist (IL1ra) and IL6 (p=0.0001) had already dropped within 24 hours and this persisted up to day 14. Systemic levels of TNFα mRNA were low and remained unchanged, though total TNFα (free and bound) in the circulation increased after D2E7, probably reflecting the presence of TNF-antiTNF mAb complexes (p<0.005, at days 1 and 14). Both TNF receptors dropped below baseline levels at day 14 (p<0.005). Despite clinical improvement of arthritis, no consistent immunohistological changes were seen two weeks after anti-TNF administration. Endothelial staining for IL1β tended to decrease in treated patients (p=0.06) but not in responders. The staining for IL1β and TNFα in sublining layers and vessels was mutually correlated (rs=0.47 and 0.58 respectively, p<0.0005) and the microscopic scores for inflammation correlated with sublining TNFα and IL1β scores (rs=0.65 and 0.54 respectively, p<0.0001), though none of these showed significant changes during the study.
CONCLUSIONS—Blocking TNFα in RA results in down regulation of IL1β mRNA at the systemic level and in reduction of the endogenous antagonists for IL1 and TNF and of other cytokines related to the acute phase response, such as IL6, within days. At the synovial level, anti-TNF treatment does not modulate IL1β and TNFα in the short term. The synovial expression of these cytokines does not reflect clinical response to TNF neutralisation.


doi:10.1136/ard.60.7.660
PMCID: PMC1753753  PMID: 11406520
19.  Influences of gamma interferon on synovial fibroblast-like cells. Ia induction and inhibition of collagen synthesis. 
Journal of Clinical Investigation  1985;76(2):837-848.
The shape and function of adherent cells cultured from rheumatoid synovial membranes are influenced by immune cells, and their products. The synovial cells produce collagenase and prostaglandin E2 (PGE2), the levels of which are increased when the cells are incubated with the monokine, mononuclear cell factor/interleukin 1. The majority of adherent synovial cells are fibroblastlike in appearance and synthesize collagens and fibronectin; the synthesis of collagens and fibronectins are also increased by a monocyte factor. In the present study we found that the fibroblastlike cells expressed major histocompatibility complex class II (Ia-like) antigens after initial dispersion from the synovial membrane. Monocyte lineage antigens were detected on some round cells in early passage, but no T lymphocytes were identified in established cultures. There was loss of Ia expression on the fibroblastlike cells with age and passage in culture. The addition of the lymphokine, gamma interferon (recombinant), induced class II antigen (DR and DS/DQ) expression in early or late passage cells in a time- and dose-dependent manner and required protein synthesis. Furthermore, the adherent synovial fibroblastlike cells continued to be Ia-positive when examined as long as 10 d after the removal of gamma interferon. Ia expression was also induced by gamma interferon in normal skin fibroblasts. Synovial cells that could be induced to express Ia also bound a monoclonal antibody to type III collagen (a fibroblast marker). Gamma interferon, while inducing Ia expression, decreased the binding of type III collagen antibody on unstimulated as well as monokine-stimulated cells. Analysis of [3H]proline-labeled medium by SDS polyacrylamide gel electrophoresis showed that gamma interferon decreased the synthesis of type I and III collagens and fibronectin by adherent synovial cells in a dose-dependent manner. These findings suggest that Ia expression by synovial tissue cells is not cell-specific, but reflects one or several related events, such as the degree of T lymphocyte infiltration, the presence of factors that stimulate gamma interferon release, or an increased sensitivity of the cells to gamma interferon. Whereas the synthesis of class II antigens is enhanced by the lymphokine gamma interferon, and a monocyte factor(s) stimulates collagen, collagenase and PGE2 synthesis by the same cells, gamma interferon inhibits basal and monokine-induced collagen synthesis. Thus, lymphokines and monokines may influence the extent of fibrosis as contrasted to matrix destruction at various stages of the rheumatoid lesion by affecting the function of fibroblastlike synovial cells.
Images
PMCID: PMC423913  PMID: 2993365
20.  Proinflammatory role of amphiregulin, an epidermal growth factor family member whose expression is augmented in rheumatoid arthritis patients 
Background
The epidermal growth factor (EGF) and EGF receptor (EGFR) families play important roles in the hyperplastic growth of several tissues as well as tumor growth. Since synovial hyperplasia in rheumatoid arthritis (RA) resembles a tumor, involvement of the EGF/EGFR families in RA pathology has been implied. Although several reports have suggested that ErbB2 is the most important member of the EGFR family for the synovitis in RA, it remains unclear which members of the EGF family are involved. To clarify the EGF-like growth factors involved in the pathology of RA, we investigated the expression levels of seven major EGF-like growth factors in RA patients compared with those in osteoarthritis (OA) patients and healthy control subjects.
Methods
The expression levels of seven EGF-like growth factors and four EGFR-like receptors were measured in mononuclear cells isolated from bone marrow and venous blood, as well as in synovial tissues, using quantitative RT-PCR. Further evidence of gene expression was obtained by ELISAs. The proinflammatory roles were assessed by the growth-promoting and cytokine-inducing effects of the corresponding recombinant proteins on cultured fibroblast-like synoviocytes (FLS).
Results
Among the seven EGF-like ligands examined, only amphiregulin (AREG) was expressed at higher levels in all three RA tissues tested compared with the levels in OA tissues. The AREG protein concentration in RA synovial fluid was also higher than that in OA synovial fluid. Furthermore, recombinant human AREG stimulated FLS to proliferate and produce several proinflammatory cytokines, including angiogenic cytokines such as interleukin-8 and vascular endothelial growth factor (VEGF), in a dose-dependent manner. The VEGF mRNA levels in RA synovia and VEGF protein concentrations in RA synovial fluid were significantly higher than those in the corresponding OA samples and highly correlated with the levels of AREG.
Conclusion
The present findings suggest that AREG functions to stimulate synovial cells and that elevated levels of AREG may be involved in the pathogenesis of RA.
doi:10.1186/1476-9255-5-5
PMCID: PMC2396620  PMID: 18439312
21.  Cytokine expression in synovial membranes of patients with rheumatoid arthritis and osteoarthritis. 
Annals of the Rheumatic Diseases  1993;52(12):870-875.
OBJECTIVES--To compare, by immunohistochemistry, the cellular and cytokine profile in rheumatoid arthritis (RA) and osteoarthritis (OA) synovial membranes (SMs). Synovium was obtained at knee arthroplasty from 10 patients with RA and 10 with OA. METHODS--Synovial membranes were stained with a panel of monoclonal antibodies (MAb) to assess cytokine expression (IL-1 alpha, IL-1 beta, IL-6, GM-CSF, TNF-alpha and EGF) and the intensity of the mononuclear cellular infiltrate (MNC). RESULTS--Significantly greater percentages of IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, GM-CSF and EGF cells were detected in all areas of the rheumatoid SMs when compared with osteoarthritic SMs. Five RA but only one OA SM demonstrated focal lymphoid aggregates. Lining layer thickening was noted in RA SMs only. The intensity of the MNC and number of blood vessels were greater in the RA group. CONCLUSION--The results suggest that the differences in cytokine production by RA and OA SMs are quantitative but that the greater thickness of the synovial lining layer and higher vascularity may be specific to RA.
Images
PMCID: PMC1005218  PMID: 8311538
22.  Measurement of Inflammatory Biomarkers in Synovial Tissue Extracts by Enzyme-Linked Immunosorbent Assay 
We developed methods for measuring inflammatory biomarkers (cytokines, chemokines, and metalloproteinases) in synovial biopsy specimens from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Soluble extracts of synovial fragments were prepared with mild detergent and analyzed by enzyme-linked immunosorbent assay (ELISA) for interleukin 1β (IL-1β), IL-6, IL-8, tumor necrosis factor alpha (TNF-α), and matrix metalloproteinase 3. The optimal detergent was 0.1% Igepal CA-630, which interfered minimally with ELISA detection but extracted 80% of IL-6 from synovial tissue. Upon spiking, 81 to 107% of added biomarkers could be recovered. To determine within-tissue variability, multiple biopsy specimens from each RA synovial extract were analyzed individually. A resulting coefficient of variation of 35 to 62% indicated that six biopsy specimens per synovial extract would result in a sampling error of ≤25%. Preliminary power analysis suggested that 8 to 15 patients per group would suffice to observe a threefold difference before and after treatment in a serial biopsy clinical study. The previously described significant differences in IL-1β, IL-6, IL-8, and TNF-α levels between RA and OA could be detected, thereby validating the use of synovial extracts for biomarker analysis in arthritis. These methods allow monitoring of biomarker protein levels in synovial tissue and could potentially be applied to early-phase clinical trials to provide a preliminary estimate of drug efficacy.
doi:10.1128/CDLI.10.6.1002-1010.2003
PMCID: PMC262451  PMID: 14607859
23.  Cytokines in chronic inflammatory arthritis. IV. Granulocyte/macrophage colony-stimulating factor-mediated induction of class II MHC antigen on human monocytes: a possible role in rheumatoid arthritis 
Granulocyte/macrophage CSF (GM-CSF) has recently been identified in rheumatoid arthritis (RA) synovial effusions. To study a potential role for GM-CSF and other cytokines on the induction of HLA-DR expression on monocytes and synovial macrophages, we analyzed the relative ability of recombinant human cytokines to induce the surface expression of class II MHC antigens on normal peripheral blood monocytes by FACS analysis. GM-CSF (800 U/ml) (mean fluorescence channel 2.54 +/- 0.33 times the control, p less than 0.001) and IFN-gamma (100 U/ml) (5.14 +/- 0.60, p less than 0.001) were the most potent inducers of HLA-DR. TNF-alpha and IL-4 also increased HLA-DR expression, although to a lesser degree [1.31 +/- 0.06 (p less than 0.02) and 1.20 +/- 0.03 (p less than 0.01), respectively]. IL-1 (40 U/ml), IL-2 (10 ng/ml), IL-3 (50 U/ml), IL-6 (100 U/ml), and CSF-1 (1,000 U/ml) did not affect surface HLA-DR density. GM-CSF also increased HLA-DR mRNA expression and surface HLA- DQ expression, but decreased CD14 (a monocyte/macrophage antigen) expression. The effect of GM-CSF on HLA-DR was not mediated by the generation of IFN-gamma in vitro because it was not blocked by anti-IFN- gamma mAb. GM-CSF was additive with IL-4 and low amounts (less than 3 U/ml) of IFN-gamma and synergistic with TNF-alpha. Because we have recently reported that supernatants of cultured RA synovial cells produce a non-IFN-gamma factor that induces HLA-DR on monocytes, we then attempted to neutralize this factor with specific anti-GM-CSF mAb. Four separate synovial tissue supernatants were studied, and the antibody neutralized the HLA-DR-inducing factor in each (p less than 0.01).
PMCID: PMC2189430  PMID: 2504878
24.  Expression and localisation of the new metalloproteinase inhibitor RECK (reversion inducing cysteine-rich protein with Kazal motifs) in inflamed synovial membranes of patients with rheumatoid arthritis 
Annals of the Rheumatic Diseases  2004;64(3):368-374.
Objective: To assess the expression and localisation of the new metalloproteinase inhibitor RECK, an inhibitor of matrix metalloproteinase-14 (MMP-14) secretion and activity, in the synovial membrane of patients with rheumatoid arthritis (RA).
Methods: RECK expression in synovium samples from patients with RA, osteoarthritis (OA), and "trauma" were studied by quantitative real time reverse transcription-polymerase chain reaction (Q-PCR). RECK mRNA levels were compared with those of the enzyme MMP-14. RECK expression on cryostat sections of synovium was disclosed by goat-antihuman RECK monoclonal antibody. RECK protein was detected on synovial cryostat sections and measured by western blotting. RECK expression on macrophages was investigated by double staining of CD68 and RECK on cryostat sections and characterised by confocal microscopy. RECK expression on RA monocytes or normal monocytes was further investigated by FACS analysis.
Results: RECK expression in the synovial membrane of patients with RA was significantly lower than in OA and controls. MMP-14 mRNA levels were not significantly different between the three groups. In RA synovium, RECK protein was expressed mainly in the lining layer but also by macrophages around blood vessels. Fibroblasts and about 50% of the CD68 positive macrophages expressed RECK. In CD68 positive macrophages, RECK was only expressed in secretory granules and not on the membrane. The same pattern was found in M-CSF cultured macrophages of patients with RA and controls. In contrast, synovial fibroblasts showed a diffuse membrane expression within the synovium similar to cultured RA fibroblasts. RECK expression was low on the membrane of monocytes according to FACS analysis.
Conclusion: The new MMP inhibitor RECK is expressed in synovial membranes of RA, OA, and controls. RECK mRNA is lowest in RA synovial membranes. In contrast with fibroblasts, macrophages in the synovium express RECK only cytoplasmically and not on their membrane.
doi:10.1136/ard.2004.027870
PMCID: PMC1755425  PMID: 15485996
25.  Angiopoietin-1 is expressed in the synovium of patients with rheumatoid arthritis and is induced by tumour necrosis factor α 
Annals of the Rheumatic Diseases  2003;62(2):100-107.
Objectives: To examine the potential role of the angiogenic growth factor angiopoietin-1 (Ang-1) in inflammatory arthritis.
Methods: Eighteen synovial tissue samples were obtained from 17 patients with a clinical diagnosis of rheumatoid arthritis (RA) and compared with six synovial tissue samples from six patients with osteoarthritis (OA). Ang-1 expression in synovial tissues was determined by immunohistochemistry and in situ hybridisation. Ang-1 mRNA and protein expression were also examined by northern blot analysis and enzyme linked immunosorbent assay (ELISA) in cultured synovial fibroblasts and human umbilical vein endothelial cells (HUVECs) before and after treatment with tumour necrosis factor (TNF)α.
Results: Ang-1 protein expression was detected by immunohistochemistry in 16/18 RA synovial tissue samples. Ang-1 protein was frequently observed in the synovial lining layer and in cells within the sublining synovial tissue, in both perivascular areas and in areas remote from vessels. In contrast, Ang-1 was only weakly detected in these sites in OA samples. Ang-1 mRNA and protein were also expressed in cultured synovial fibroblasts derived from patients with RA. In addition, induction of Ang-1 mRNA and protein was observed by northern blot analysis and ELISA after stimulation of RA synovial fibroblasts, but not HUVECs, with the proinflammatory cytokine TNFα.
Conclusions: Ang-1 mRNA and protein are expressed in the synovium of patients with RA, and are up regulated in synovial fibroblasts by TNFα. Ang-1 may therefore be an important regulator of angiogenesis in inflammatory arthritis.
doi:10.1136/ard.62.2.100
PMCID: PMC1754433  PMID: 12525377

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