Cells In Rheumatoid Arthritis Synovial Tissue Research Articles

CD14+ Dendritic-Shaped Cells Functioning as Dendritic Cells in Rheumatoid Arthritis Synovial Tissues.

We previously reported that CD14+ dendritic-shaped cells exhibit a dendritic morphology, engage in pseudo-emperipolesis with lymphocytes, and express CD90 in the perivascular areas of rheumatoid arthritis (RA) synovial tissues. However, it remains unclear whether these CD14highCD90intermediate(int) cells function as dendritic cells. In this study, we investigated the dendritic cell-differentiation potential of CD14highCD90int cells. The localization and number of CD14highCD90int cells in RA synovial tissues and peripheral blood were examined. The dendritic cell-differentiation potential of CD14highCD90int cells was examined by measuring interleukin-6 and tumor necrosis factor-α levels in the supernatant and CD83 and human leukocyte antigen (HLA)-DR expression in the cells after induction of dendritic cell differentiation. Synovial cells were co-cultured with lymphocytes, and the activation of these cells was examined. CD14highCD90int cells were abundant in RA synovial tissues, including the sublining layer and the pannus areas. Patients with untreated and active RA had significantly higher percentages of CD14highCD90int cells in the peripheral blood and synovial tissues. In RA synovial cells, inflammatory cytokine levels increased with dendritic cell-differentiation culture, but CD83 and HLA-DR expression were significantly increased in the CD14highCD90int cell group. When co-cultured with lymphocytes, cell numbers and inflammatory cytokine levels significantly increased in both groups of synovial cells after dendritic cell induction. CD14+ cells migrate and spread from the circulating blood to RA synovial tissues while expressing CD90, and CD14highCD90int cells in contact with lymphocytes differentiate into HLA-DR+ dendritic cells, which contribute to chronic inflammation in RA.

Identification and preliminary validation of synovial tissue-specific genes and their-mediated biological mechanisms in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease. It is well known that the formation of positive feedback between synovial hyperplasia and inflammatory infiltration is intimately associated with the occurrence and development of RA. However, the exact mechanisms still remain unknown, making the early diagnosis and therapy of RA difficult. This study was designed to identify prospective diagnostic and therapeutic biomarkers, as well as their-mediated biological mechanisms in RA. Three microarray datasets (GSE36700, GSE77298 and GSE153015) and two RNA-sequencing datasets (GSE89408 and GSE112656) of synovial tissues, as well as three other microarray datasets (GSE101193, GSE134087 and GSE94519) of peripheral blood were downloaded for integrated analysis. The differently expressed genes (DEGs) were identified by "limma" package of R software. Then, weight gene co-expression analysis and gene set enrichment analysis were performed to investigate synovial tissue-specific genes and their-mediated biological mechanisms in RA. The expression of candidate genes and their diagnostic value for RA were verified by quantitative real-time PCR and receiver operating characteristic (ROC) curve, respectively. Relevant biological mechanisms were explored through cell proliferation and colony formation assay. The suggestive anti-RA compounds were discovered by CMap analysis. We identified a total of 266 DEGs, which were mainly enriched in cellular proliferation and migration, infection and inflammatory immune signaling pathways. Bioinformatics analysis and molecular validation revealed 5 synovial tissue-specific genes, which exhibited excellent diagnostic value for RA. The infiltration level of immune cells in RA synovial tissue was significantly higher than that in control individuals. Moreover, preliminary molecular experiments suggested that these characteristic genes may be responsible for the high proliferation potential of RA fibroblast-like synoviocytes (FLSs). Finally, 8 small molecular compounds with anti-RA potential were obtained. We have proposed 5 potential diagnostic and therapeutic biomarkers (CDK1, TTK, HMMR, DLGAP5, and SKA3) in synovial tissues that may contribute to the pathogenesis of RA. These findings may shed light on the early diagnosis and therapy of RA.

The growth factor midkine may play a pathophysiological role in rheumatoid arthritis

Objectives: Midkine (MK) is involved in cell proliferation, differentiation, migration, and survival. In this study, we measured serum MK levels in rheumatoid arthritis (RA) and investigated the correlation of serum MK with RA disease activity. Expression and effect of MK in RA synovial tissue were also examined.Methods: Serum MK and production of inflammatory mediators by rheumatoid synovial fibroblasts (RSFs) were measured by enzyme-linked immunosorbent assay. MK expression in synovial tissue was examined by immunohistochemistry. MK receptor expression was analyzed by RT-PCR and Western blotting.Results: RA patients had a significantly higher serum MK level than healthy controls. In RA patients, the MK level was correlated with DAS28-ESR, disability index of the Health Assessment Questionnaire, and rheumatoid factor level. The serum MK level tended to be decreased by anti-TNF therapy. MK was expressed by synovial lining cells in RA synovial tissues and it enhanced the production of IL-6, IL-8, and CCL2 by RSFs. RSFs expressed LDL receptor-related protein 1, candidate receptor for MK.Conclusions: The serum MK level could be a marker of disease activity in RA and an indicator of a poor prognosis. MK may have a role in the pathogenesis of RA via induction of inflammatory mediators.

Acute serum amyloid A regulates cytoskeletal rearrangement, cell matrix interactions and promotes cell migration in rheumatoid arthritis

ObjectiveSerum amyloid A (A-SAA) is an acute-phase protein with cytokine-like properties implicated in the pathogenesis of rheumatoid arthritis (RA), atherosclerosis, diabetes and Alzheimer's disease. This study characterises the mechanism of...

Involvement of breast cancer resistance protein expression on rheumatoid arthritis synovial tissue macrophages in resistance to methotrexate and leflunomide

To determine whether multidrug-resistance efflux transporters are expressed on immune effector cells in synovial tissue from patients with rheumatoid arthritis (RA) and compromise the efficacy of methotrexate (MTX) and leflunomide (LEF). Synovial tissue biopsy samples obtained from RA patients before treatment and 4 months after starting treatment with MTX (n = 17) or LEF (n = 13) were examined by immunohistochemical staining and digital image analysis for the expression of the drug efflux transporters P-glycoprotein, multidrug resistance-associated protein 1 (MRP-1) through MRP-5, MRP-8, MRP-9, and breast cancer resistance protein (BCRP), and the relationship to clinical efficacy of MTX and LEF was assessed. BCRP expression was observed in all RA synovial biopsy samples, both pretreatment and posttreatment, but not in control noninflammatory synovial tissue samples from orthopedic patients. BCRP expression was found both in the intimal lining layer and on macrophages and endothelial cells in the synovial sublining. Total numbers of macrophages in RA patients decreased upon treatment; in biopsy samples with persistently high macrophage counts, 2-fold higher BCRP expression was observed. Furthermore, median BCRP expression was significantly increased (3-fold) in nonresponders to disease-modifying antirheumatic drugs (DMARDs) compared with responders to DMARDs (P = 0.048). Low expression of MRP-1 was found on synovial macrophages, along with moderate expression in T cell areas of synovial biopsy specimens from one-third of the RA patients. These findings show that the drug resistance-related proteins BCRP and MRP-1 are expressed on inflammatory cells in RA synovial tissue. Since MTX is a substrate for both BCRP and MRP-1, and LEF is a high-affinity substrate for BCRP, these transporters may contribute to reduced therapeutic efficacy of these DMARDs.

In vitro and in vivo efficacy of a recombinant immunotoxin against folate receptor β on the activation and proliferation of rheumatoid arthritis synovial cells

To investigate the effects of the recombinant immunotoxin dsFv anti-FRbeta-PE38, which consists of the disulfide-stabilized Fv fragment (dsFv) of the anti-folate receptor beta (anti-FRbeta) antibody and the 38-kd portion of Pseudomonas exotoxin A (PE38), on the activation and proliferation of cells that function in inflammatory and degradative processes in rheumatoid arthritis (RA) synovial tissue. The Ig VH-PE38 fusion protein and the Ig VL protein were produced in Escherichia coli, and then joined with a disulfide bond by engineering cysteine residues in the framework regions of these proteins. The effects of dsFv anti-FRbeta-PE38 on the activation and proliferation of cells in RA synovial tissue were investigated by immunohistochemistry; the numbers of cells expressing CD68, vascular cell adhesion molecule 1, angiopoietin 1, CD34, proliferating cell nuclear antigen, and interleukin-6 and the numbers of apoptotic cells were counted in RA synovial tissue engrafted into SCID mice treated or not treated with dsFv anti-FRbeta-PE38. The effects of dsFv anti-FRbeta-PE38 on the generation of osteoclasts from RA adherent synovial mononuclear cells in vitro was investigated by counting the number of resorption pits on dentin slices treated or not treated with dsFv anti-FRbeta-PE38. Administration of dsFv anti-FRbeta-PE38 reduced the numbers of macrophages, activated fibroblast-like cells, endothelial cells, and proliferating cells and increased the numbers of apoptotic cells in RA synovial tissue engrafted into SCID mice. In vitro, the generation of osteoclasts from RA adherent synovial mononuclear cells was largely suppressed by treatment with dsFv anti-FRbeta-PE38. Our findings show that dsFv anti-FRbeta-PE38 immunotoxin would be a promising tool for the treatment of RA synovitis, especially when administered intraarticularly.

Effects of antirheumatic treatments on the prostaglandin E2 biosynthetic pathway

Microsomal prostaglandin E synthase 1 (mPGES-1) is up-regulated in experimental arthritis and markedly expressed in synovial tissue biopsy samples from patients with rheumatoid arthritis (RA). This study was carried out to determine the effects of tumor necrosis factor (TNF) blockers and glucocorticoids on mPGES-1 and cyclooxygenase (COX) expression, as well as biosynthesis of PGE(2) in rheumatoid joints. In vitro effects of TNF blockers and dexamethasone on the PGE(2) biosynthetic pathway were examined in RA synovial fluid mononuclear cells (SFMCs) by flow cytometry. PGE(2) levels in culture supernatants were measured by enzyme immunoassay. Expression of enzymes responsible for PGE(2) synthesis ex vivo was evaluated by immunohistochemistry in synovial biopsy samples obtained from 18 patients before and after treatment with TNF blockers and from 16 patients before and after intraarticular treatment with glucocorticoids. Double immunofluorescence was performed using antibodies against mPGES-1, COX-1, COX-2, and CD163. Double immunofluorescence revealed that mPGES-1 and COX-2 were colocalized in SFMCs as well as in RA synovial tissue cells. The addition of either TNF blockers or dexamethasone suppressed lipopolysaccharide-induced mPGES-1 and COX-2 expression in synovial fluid monocyte/macrophages in vitro and decreased the production of PGE(2). Intraarticular treatment with glucocorticoids significantly reduced both mPGES-1 and COX-2 expression in arthritic synovial tissue ex vivo. The number of COX-1-expressing cells in synovial tissue was also significantly decreased by glucocorticoid treatment. In contrast, neither mPGES-1 nor COX-2 expression in synovial tissue was significantly suppressed by anti-TNF therapy. These data are the first to demonstrate the effects of antirheumatic treatments on mPGES-1 expression in RA and suggest that the inhibition of PGE(2) biosynthesis, preferably by targeting mPGES-1, might complement anti-TNF treatment for optimal antiinflammatory results in RA.

The effects of inflammatory arthritis on bone remodeling

Rheumatoid arthritis (RA) represents an excellent model for gaining insights into the adverse effects of inflammatory arthritis on skeletal remodeling. Bone erosions at the margins of diarthrodial joints represent the radiographic hallmark of RA. Histopathologic examination of the bone–pannus interface in patients with RA reveals the presence of a heterogeneous population of cells lining the bone surface, including multinucleated cells expressing the full phenotypic repertoire of osteoclasts, the cells that mediate bone resorption in physiologic remodeling. Further evidence implicating osteoclasts in the pathogenesis of marginal joint erosions is derived from animal models of inflammatory arthritis in which animals lacking the capacity to form osteoclasts fail to develop joint erosions. Studies have shown that the RA synovial tissue contains abundant osteoclast precursors and that the RA synovium is a rich source of factors with potent osteoclast differentiation and activation activity. Particular attention has focused on receptor activator of NF-κB ligand (RANKL), a member of the tumor necrosis factor ligand family, because of the requirement of this factor for osteoclastogenesis. RANKL is expressed by synovial fibroblasts and activated T cells in RA synovial tissues and in several different animal models of inflammatory arthritis, treatment with osteoprotegerin (the soluble receptor that inhibits RANKL activity) results in marked suppression of focal bone erosions. In addition, mice possessing the null mutation for RANKL are protected from focal bone destruction in the serum transfer model of inflammatory arthritis. An additional observation in patients with active RA is the absence of radiographic evidence of bone repair. This finding suggests that the processes that regulate coupling of bone resorption and formation under physiologic conditions have been disrupted and that the enhanced focal bone resorption is not matched by a compensatory increase in bone formation. In addition to the disordered focal bone remodeling associated with the synovial lesion, patients with RA also exhibit evidence of generalized axial and appendicular bone loss at sites that are distant from inflamed joints. Assessment of biochemical markers of bone turnover indicates that there is a generalized increase in bone resorption, and that there is a correlation between disease activity and the rate of systemic bone resorption. It is likely that this generalized skeletal bone loss is mediated by synovial cytokines with osteoclastogenic activity that enter the circulation and act as 'endocrine' factors to adversely affect systemic bone remodeling. Recent studies indicate that agents, such as the bisphosphonates, that target osteoclast-mediated bone resorption have the capacity to reduce not only generalized bone osteoporosis but also may have efficacy in retarding the progression of focal joint erosions.

Regulation of interleukin-18 binding protein production by blood and synovial cells from patients with rheumatoid arthritis.

To study the regulation of interleukin-18 binding protein (IL-18BP) production by rheumatoid arthritis (RA) or control peripheral blood mononuclear cells (PBMCs) and by RA synovial tissue cells, and to compare the levels of IL-18BP messenger RNA (mRNA) expression in whole blood from RA patients and controls. Unstimulated or phytohemagglutinin and phorbol myristate acetate (PHA/PMA)-stimulated PBMCs from 10 RA patients and 12 healthy controls and unstimulated or PHA/PMA-stimulated synovial tissue cells from 8 RA patients were cultured with or without IL-12 (1 ng/ml) and IL-18 (5 ng/ml) alone or in combination. IL-18BP and interferon-gamma (IFN gamma) levels in supernatants were measured by enzyme-linked immunosorbent assay. Levels of IL-18BP and IFN gamma mRNA expression in whole blood samples from 22 RA patients and 12 healthy controls were determined by quantitative reverse transcriptase-polymerase chain reaction. IL-12 decreased the basal levels of IL-18BP production by freshly isolated RA or control PBMCs, but increased those of synovial cells or PHA/PMA-stimulated PBMCs. IL-18 alone had no direct effect on IL-18BP production by PBMCs or RA synovial cells, with or without stimulation. Unstimulated whole blood samples from RA patients showed lower levels of IL-18BP mRNA expression than those from healthy controls. The production of IL-18BP in response to IL-12 and IL-18 was regulated differently in blood and synovial cells. The difference appears to be related to the level of cell activation.

Adverse effects of rheumatoid arthritis on bone remodeling

Rheumatoid arthritis (RA) represents an excellent model for gaining insights into the adverse effects of inflammatory arthritis on local articular as well as generalized systemic bone remodeling. Bone loss manifested by focal erosions at the margins of diarthrodial joints represents the radiographic hallmark of RA. These lesions are produced by resorption of cortical bone at the bone–pannus junction. Inflammatory pannus can also extend into the marrow space, with accompanying subcortical and trabecular bone destruction. In animal models of inflammatory arthritis, erosion of subchondral bone contributes significantly to cartilage loss, as the scaffolding bone is destroyed by the inflammatory process. Preservation of subchondral bone integrity would be predicted to have a cartilage sparing effect even in the presence of continued intra-articular joint inflammation. Recent studies employing magnetic resonance imaging have shown that marginal joint erosions occur very early in the course of RA and progress throughout the disease [1,2]. The propensity of the inflamed pannus tissue in RA to induce bone resorption is probably related to its capacity to produce a variety of factors with potent osteoclast differentiation and activation activity. Particular attention has focused on receptor activator of NF-κB ligand (RANKL), a member of the tumor necrosis factor ligand family, because of the requirement of this factor for osteoclastogenesis. RANKL is expressed by synovial fibroblasts and activated T cells in RA synovial tissues [3]. In three different animal models of inflammatory arthritis, treatment with osteoprotegerin (the soluble receptor that inhibits RANKL activity) results in marked suppression of focal bone erosions [4-6]. In addition, mice possessing the null mutation for RANKL are protected from focal bone destruction in the serum transfer model of inflammatory arthritis [7]. These observations lend support to the concept that enhanced osteoclast-mediated bone resorption at the pannus–bone interface and in subchondral and trabecular bone play a critical role in the pathogenesis of focal articular bone erosions. An additional observation in patients with active RA is the absence of bone repair radiographically. This finding suggests that the processes that regulate coupling of bone resorption and formation under physiologic conditions have been disrupted, and that the enhanced focal bone resorption associated with the synovial inflammatory lesion is not matched by a compensatory increase in bone formation. Of particular interest will be the determination of the effects of therapies that inhibit joint erosions on these focal bone remodeling events at the bone–pannus interface and in the subchondral bone. In addition to the disordered focal bone remodeling associated with the synovitis, patients with RA also exhibit evidence of generalized axial and appendicular osteopenia at sites that are distant from inflamed joints [8]. The reduction in bone mass has been confirmed using multiple different techniques, and patients with RA have an increased risk of hip and vertebral fractures [9]. Assessment of biochemical markers of bone turnover indicates that there is a generalized increase in bone resorption, and that there is a correlation between disease activity and the rate of systemic bone resorption. Patients with greater disease activity exhibit enhanced rates of bone loss. It is likely that the disturbance in systemic bone remodeling is mediated by proinflammatory cytokines with osteoclastogenic activity that are released into the circulation from the inflamed joints. These factors probably then act systemically to produce a generalized increase in osteoclast-mediated bone resorption. Bisphosphonates have been shown to reverse systemic bone loss in patients with RA, but studies thus far have not shown that these treatment regimens reduce the progression of focal bone erosions [10]. It is likely, however, that new approaches for more effectively inhibiting osteoclast-mediated bone resorption will become available; for example, agents that specifically inhibit osteoclast formation or activity by targeting mediators such as RANKL. Whether preservation of the skeletal architecture, independent of, or in addition to suppression of joint and systemic inflammation, will impact on the progression of functional disability needs to be investigated in appropriately designed clinical trials.

Effect of IL-18 on synovial cell subsets and its regulation by IL-18BP

IL-18 has multiple biological activities that are important in generating Th1 responses and inflammatory tissue damage. IL-18 has molecular similarity with IL-1, and IL-18 receptor (IL-18R) belongs to the superfamily of IL-1 receptor. In our previous studies, IL-18 was detected in both synovial fluid and serum samples from patients with rheumatoid arthritis (RA) and serum IL-18 levels correlated with disease activity as assessed by levels of serum CRP and erythrocyte sedimentation rate. We then examined the synovial expression of IL-18 in RA. Isolated RA tissue cells could spontaneously produce high amounts of IL-18 in culture. In RA synovium, IL-18-positive cells were frequently located in both the lining and sublining layer, but not in lymphocyte aggregates. Both CD14+ macrophages and synoviocytes isolated from RA synovium expressed detectable levels of IL-18 mRNA. Using IL-1 as a positive control, we next examined the effect of IL-18 on IL-6 production in RA synoviocyte culture. In contrast to IL-1, IL-18 has no effect on IL-6 production from RA synoviocyte at both mRNA and protein levels. This lack of response appears to be related to defective receptor expression. Isolated RA tissue cells as well as nonstimulated RA synoviocytes expressed detectable levels of mRNA of IL-18R alpha chain, but IL-18R beta chain, which is critical for IL-18 signal transduction, was detected in RA tissue cells but not in purified synoviocytes, even if these cells were stimulated with IL-1β, TNF-α, or supernatant of RA synovium culture. RA synovial cells responded to IL-18. IFN-γ production by IL-12-stimulated RA synovial tissue cells was synergistically enhanced by IL-18, and it was partially inhibited by addition of IL-18 binding protein (IL-18BP). Upregulation of IL-18R beta mRNA levels in RA synovial tissue cells with IL-12 stimulation could explain this synergy. These results indicate a complex effect of IL-18 on various cell subsets found in RA synovium. IL-18 appears to further enhance the Th1 promoting activity of IL-12, an effect which is regulated by IL-18BP.

Selective recruitment of CCR6-expressing cells by increased production of MIP-3 alpha in rheumatoid arthritis.

Infiltration of various types of leucocytes has been shown to play a crucial role in the pathogenesis of rheumatoid arthritis (RA). Macrophage inflammatory protein-3 alpha (MIP-3 alpha) is a recently identified chemokine which is a selective chemoattractant for leucocytes such as memory T cells, naïve B cells and immature dendritic cells. In this study, we investigated the expression of MIP-3 alpha and its specific receptor CCR6 in the inflamed joints of patients with RA. Increased amounts of MIP-3 alpha were found by ELISA in synovial fluids (SF) of patients with RA. MIP-3 alpha was apparently detected in all synovial tissue specimens of RA patients (n = 6), but it could not be detected in that of osteoarthritis (OA) patients (n = 4). Expression of MIP-3 alpha was detected especially in the sublining layer, and infiltrating mononuclear cells in RA synovial tissue. Gene expression of MIP-3 alpha was also found in six out of 11 RA-synovial fluid cells by RT-PCR. Cultured synovial fibroblasts derived from either RA or OA patients were capable of producing MIP-3 alpha in response to IL-1 beta and TNFalpha in vitro. Furthermore, expression of CCR6 was found in infiltrating mononuclear cells in the cellular clusters and around the vessels of RA synovial tissue. These findings indicate that increased production of MIP-3 alpha may contribute to the selective recruitment of CCR6-expressing cells in RA.

Intracellular expression of CXCR3 on rheumatoid arthritis synovial tissue cells

Inflammatory cell infiltration and synovial activation are important processes in rheumatoid arthritis. Chemotactic gradients of various chemokines are responsible for cell attraction and possibly for their activation. We have previously detected strong expression of chemokine receptor CXCR3 in the rheumatoid joint by immunostaining.

Interferon-?-inducing activity of interleukin-18 in the joint with rheumatoid arthritis

To examine the levels of interleukin-18 (IL-18) bioactivity within the rheumatoid arthritis (RA) joint, and the differential effects of IL-12 and IL-18 on interferon-gamma (IFNgamma) production by T cell infiltrates. Expression of IL-18 protein and messenger RNA (mRNA) was determined by enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction, respectively. The biologic activity of IL-18 was detected on the basis of IFNgamma secretion from IL-18-responding human myelomonocytic KG-1 cells. To determine the extent of inhibitory activity on binding of IL-18 to its receptor, a [125I]-IL-18 binding inhibition assay was performed, using a Chinese hamster ovary cell line transfected with a murine IL-18 receptor. The amount of IL-18 protein detected in both the serum and synovial fluid of RA patients was markedly larger than that detected in the serum and synovial fluid ofosteoarthritis (OA) patients, and serum IL-18 levels correlated with the levels of serum C-reactive protein. IFNgamma production by KG-1 cells was more strongly stimulated in synovial fluid samples from RA patients than in samples from OA patients, and this activity was largely diminished in the presence of anti-IL-18 antibody. In contrast, the activity of IL-18 binding inhibition in the serum and synovial fluid of RA patients was not significantly elevated compared with that in OA patients. RA synovial tissues showed increased expression of IL-18 mRNA and increased IL-18 protein synthesis compared with that in OA tissues. Purified CD14+ macrophages, but not activated fibroblast cell lines, from RA synovium were able to release mature IL-18, although both cell types expressed its transcripts. IL-18 alone showed a negligible effect on IFNgamma production by RA synovial tissue cells, in contrast to IL-12, which was directly stimulatory. However, IL-12-induced IFNgamma production was synergistically enhanced by IL-18, and yet was >50% reduced by neutralization of endogenous IL-18 with anti-IL-18 antibody. These results indicate that IL-18, produced predominantly by tissue macrophages, primarily potentiates IL-12-induced IFNgamma production by T cell infiltrates in RA synovium. Detection of significant IL-18 bioactivity in the joints, despite the presence of IL-18 binding inhibitors, supports an integral role of this cytokine in perpetuating the IFNgamma-dominant T cell cytokine response in RA.

Expression of peroxisome proliferator-activated receptorγ (PPARγ) in synovial tissues of inflammatory arthritis

Peroxisome proliferator-activated receptor γ (PPARγ) is transcription factors belong to the nuclear receptor gene family and binds to specific response elements termed PPRE, thus regulating the expression of target genes. PPARγ triggers adipocyte differentiation in adipose tissue and inhibits the cytokine production in macrophage. We analyzed the expression of PPARγ in human synovial tissues of patient with rheumatoid arthritis (RA) or osteoarthritis (OA) . PPARγ mRNA strongly expressed in synovial tissues with active RA synovitis more than that of OA. Immunohistological analysis demonstrated PPARγ localized in nuclear area of synovial lining macrophages and vascular endothelial cells in RA synovial tissues. PPARγ expression correlated with the rate of macrophage infiltration and hypervas-cularization in synovial tissues. We measured the expression of PPARγ mRNA in rat arthritic synovial tissues. PT-PCR analysis demonstrated the strong expression of PPARy in rat arthritic joints.PPARγ ligand, antidiabetic thiazolidinediones (troglitazone) inhibited the synovitis of adjuvant arthritis. These data suggest PPARγ ligands may have therapeutic application in inflammatory arthritis, such as RA.

Expression of Receptor Tyrosine Kinase Axl and its Ligand Gas6 in Rheumatoid Arthritis: Evidence for a Novel Endothelial Cell Survival Pathway

Angiogenesis and synovial cell hyperplasia are characteristic features of rheumatoid arthritis (RA). Many growth and survival factors use receptors belonging to the tyrosine kinase family that share conserved motifs within the intracellular catalytic domains. To understand further the molecular basis of cellular hyperplasia in RA, we have used degenerate primers based on these motifs and RNA obtained from the synovium of a patient with RA to perform reverse transcriptase-polymerase chain reaction. We report detection of the receptor tyrosine kinase (RTK) Axl in RA synovium and we document the expression pattern of Axl in capillary endothelium, in vascular smooth muscle cells of arterioles and veins, and in a subset of synovial cells in RA synovial tissue. Gas6 (for growth arrest-specific gene 6), which is a ligand for Axl and is related to the coagulation factor protein S, was found in synovial fluid and tissue from patients with RA and osteoarthritis. Axl expression and function was studied in human umbilical vein endothelial cells (HUVECs). Gas6 bound to HUVECs; soluble Axl inhibited this binding. Exogenous Gas6 protected HUVECs from apoptosis in response to growth factor withdrawal and from TNFalpha-mediated cytotoxicity. These findings may reveal a new aspect of vascular physiology, which may also be relevant to formation and maintenance of the abnormal vasculature in the rheumatoid synovium.

Cytokine production by synovial T cells in rheumatoid arthritis

To investigate the production of cytokines by T cells in patients with rheumatoid arthritis (RA), reactive arthritis (REA) and osteoarthritis (OA). The lymphokines interleukin (IL)-2, IL-4, interferon gamma (IFN-gamma) and tumour necrosis factor beta (TNF-beta), as well as the monokines IL-1, IL-6 and TNF-alpha, were measured by immunoassays in sera and synovial fluid (SF) from patients with RA, REA and OA. In addition, cytokine expression was studied by immunohistochemistry in synovial membrane tissue sections from patients with RA and OA. Almost 60% of RA sera contained at least one of the cytokines investigated, though in low concentrations, whereas cytokines were generally not detectable in sera from REA and OA patients. In contrast, cytokines were found in virtually all SF; thus, the majority of SF from RA patients contained IFN-gamma (median level 17 pg/ml) in addition to the monokines IL-6 (4700 pg/ml) and TNF-alpha (157 pg/ml). IFN-gamma and IL-6 (but not TNF-alpha) were also frequently measured in SF from REA patients, whereas OA samples typically contained only IL-6. Immunohistochemical analysis of tissue sections from RA patients revealed lymphokine expression in 0.1-0.3% of T cells, particularly IL-2 and IFN-gamma, and to a lesser extent also IL-4. Interestingly, the expression of TNF-alpha and IL-6 by synovial T cells was also observed. The majority of cytokine-expressing T cells were CD4-positive T-helper cells typically found in perivascular areas, whereas cytokine-producing CD8-positive T cells were found distributed throughout the synovium. As expected, in specimens from OA patients, T cells were much less abundant and expression of cytokines could not be detected. These data clearly demonstrate production of cytokines by T cells in RA synovial tissue, indicating that activated T cells play a role in the pathophysiological events of RA.

Expression of interleukin-12 in synovial tissue from patients with rheumatoid arthritis.

To examine the importance of interleukin-12 (IL-12) as a factor in the interferon-gamma (IFNgamma)-dominant T cell cytokine response in the synovial tissue of patients with rheumatoid arthritis (RA). The expression of IL-12 in synovial tissue samples from patients with chronic RA (> or = 2 years) was compared with that in samples from osteoarthritis (OA) patients by detection of IL-12 p40 messenger RNA (mRNA) using reverse transcriptase-polymerase chain reaction, measurement of IL-12 p70 protein in culture supernatants of tissue cells by immunoassay, and immunostaining of tissue sections with anti-IL-12 p70. The production of IFNgamma by RA synovial tissue cells cultured with or without IL-12 was determined. In addition, T cells were obtained 14 days after culturing RA synovial tissue cells with IL-2 alone or with IL-2 plus IL-12, neutralizing anti-IL-12, or IL-4, and cytokine patterns (i.e., IFNgamma and IL-4 levels) were determined by stimulating cells for 24 hours with anti-CD3 plus phorbol myristate acetate. Synovial tissues from RA patients more strongly expressed IL-12 p40 mRNA than did OA tissues. Dissociated tissue cells from 21 of 37 RA patients spontaneously released detectable amounts of IL-12 p70 (> or = 12.5 pg/ml) in culture, whereas production of IL-12 by OA tissues was limited. By immunohistochemical analysis, IL-12-producing cells were localized mainly in the sublining layer of RA synovium, and mostly expressed the CD68 antigen. Levels of IFNgamma production by RA synovial tissue cells were potently and selectively enhanced by IL-12. The ability of IL-2-expanding synovial T cells to produce IFNgamma was augmented by costimulation with IL-12 and diminished by anti-IL-12, while it was not affected by IL-4. These data suggest that IL-12, produced mainly by macrophage-lineage cells, may be involved in IFNgamma-dominant cytokine production by infiltrating T cells in joints with chronic RA.

Expression of Fas Antigen and Fas Ligand in the Rheumatoid Synovial Tissue

To understand the role of apoptosis through Fas/Fas ligand (Fas-L) interaction in the pathogenesis of rheumatoid arthritis (RA), we examined the expression of Fas antigen, Fas-L, and apoptosis in synovial tissue obtained from eight patients with RA and five patients with osteoarthritis (OA). Immunohistochemical staining demonstrated the significant expression of Fas antigen and Fas-L in RA synovial tissue compared with that in OA synovial tissue. Immunohistochemical staining and the DNA nick end labeling (TUNEL) method were combined and revealed that approximately 10 to 30% of Fas antigen-expressing cells in the RA synovium showed DNA fragmentation characteristic for apoptosis. In double-staining analysis, Fas-L was expressed on up to 10% of CD45RO-, CD4-, CD8-, or CD56-positive mononuclear cells in RA synovial tissue. Our results suggest that activated T cells and natural killer cells infiltrating into the RA synovium may contribute to the induction of apoptosis of RA synovial and mononuclear cells through Fas/Fas-L interaction.

Induction of Fas-dependent apoptosis in synovial infiltrating cells in rheumatoid arthritis.

Apoptosis is a feature of the synovium of rheumatoid arthritis (RA). We have recently shown that RA synoviocytes were susceptible to anti-Fas mAb and undergo apoptosis in vitro. To investigate whether infiltrating mononuclear cells also undergo Fas-dependent apoptosis, double-labeling techniques combined with immunohistochemical examination with anti-CD3 mAb and the TdT-mediated dUTP-blotin nick end labeling (TUNEL) method to detect apoptotic cells, or in situ RT assay to detect Fas mRNA, were performed using frozen tissue sections. We also examined the in vitro induction of Fas-dependent apoptosis in freshly isolated synovium infiltrating mononuclear cells (SIM), synovial stromal cells (SSC) and peripheral blood lymphocytes (PBL) using tissues from nine patients with RA and three with osteoarthritis (OA). The results showed expression of Fas antigen and apoptotic cells in a number of CD3-bearing cells in RA synovial tissues. In vitro treatment with anti-Fas mAb produced a significant apoptosis of RA SIM and SSC, while none of PBL, and neither SIM nor SSC from OA exhibited apoptosis. Moreover, approximately 50% of CD4+, CD3+ and CD45RO+ cells, and > 90% of Fas-expressing cells of RA SIM underwent apoptosis in response to anti-Fas mAb, as detected by flow cytometry. Our results suggest that RA synovial infiltrating lymphocytes acquire high susceptibility to anti-Fas mAb and undergo apoptosis. Such a phenomenon of infiltrating T cells in RA synovium may play an important pathophysiological role and suggest a possible therapeutic effect for anti-Fas mAb in RA.