Synovial Lining Cells Research Articles

The effect of sonication on simulated osteoarthritis. Part I: Effects of 1 MHz ultrasound on uptake of hyaluronan into the rabbit synovium

High molecular weight (MW) hyaluronan (HA) preparation is considered to be more biologically active than HAs of lower MWs. However, many of the HA preparations currently used to treat osteoarthritis (OA) have lower MWs by the enhanced penetration of HA molecules into the synovial lining cells. In this study, we determined the effectiveness of sonophoresis on the delivery of high MW HA into synovial membrane using an animal model of OA. A total of 1000 kDa (HA1000) and 3000 kDa (HA3000) HA were labeled with fluorescein and injected into the knees of rabbits. Low-intensity continuous ultrasound at 1 MHz, 400 mW/cm 2 was applied to the knees for 10 min treatment bid. Synovial fluid analysis revealed increased absorption and fluorescence microscopy showed deeper penetration of both HA1000 and HA3000, more so with the latter. Histologic examination indicated that ultrasound treatment resulted in no apparent damage to the synovial membrane. These results suggest that simultaneous sonication with HA injection might compensate for the short half-life of HA. Consequently, this dual treatment would render HA a far more effective tool in the management of OA. (E-mail: bhmin@ajou.ac.kr)

Immunohistological localization of Notch receptors and their ligands Delta and Jagged in synovial tissues of rheumatoid arthritis

BackgroundThe interaction of Notch receptors with their transmembrane ligands Delta and Jagged plays an important role not only in the organization of a variety of tissues but also in several genetic disorders and cancer development. The functional involvement of the Notch signaling in rheumatoid arthritis (RA) has been reported previously, but the expression profile of Notch-related molecules, as well as their relation with clinicopathological parameters, remains unclear. MethodsIn this study, we analyzed the immunohistochemical staining pattern of four Notch receptors (Notch1–4) and their ligands (Delta1 and Jagged1) in 14 synovial tissues obtained from 14 RA patients. ResultsNotch2 and Notch4 were expressed in limited areas in a few samples or in small blood vessels, respectively. Notch1, Notch3, Delta1, and Jagged1 were overexpressed in the synovial lining and sublining cells on synovial hyperplastic lesions in all samples. Notch1 expression was also observed in T and B lymphocytes of lymphoid follicles independently. Notch1 and Notch3 expression overlapped with that of Jagged1, as determined by confocal microscopy. Activation of Notch1 signaling in the RA synovium was identified using a specific antibody to the cleaved form of Notch1. The expression of these molecules did not show any correlation with clinicopathological parameters. ConclusionsOur results suggest that Notch signaling is activated in RA synovium but does not necessarily reflect the pathological condition of RA.

Macroscopic and microscopic features of synovial membrane inflammation in the osteoarthritic knee: Correlating magnetic resonance imaging findings with disease severity

To determine the magnetic resonance imaging (MRI), macroscopic, and microscopic characteristics of synovial membrane inflammation, to study the relationship between disease severity and the degree of synovial inflammation on MRI and on macroscopic and microscopic examination, and to look for colocalization of chondral lesions and synovial inflammation. Thirty-nine patients with knee osteoarthritis (OA) were classified into 2 groups according to the severity of cartilage lesions as revealed by chondroscopy. Group 1 (n = 14) had mild cartilage lesion(s) without exposure of subchondral bone. Group 2 (n = 25) had severe cartilage lesion(s) with focal or diffuse exposure of subchondral bone. Synovitis was evaluated on T1-weighted MRI sequences according to the degree of synovial thickening on a 4-point scale (ranging from 0 to 3) in 5 regions of interest. Synovial membrane was macroscopically scored, and biopsies were performed on the 5 preselected sites for histologic scoring. The mean +/- SD synovial thickening score on MRI was 1.55 +/- 0.90, with no significant difference between groups 1 and 2. Intra- and interobserver reproducibility of the total synovial score was excellent, and interobserver reproducibility of the MRI grade was good. Synovitis was diffuse and associated with chondral lesions only in the medial femorotibial compartment (r = 0.49, P = 0.001). The degree of synovial thickening on MRI correlated with qualitative macroscopic analysis (r(s) = 0.58, P < 0.001) and with microscopic features (synovial lining cells [r(s) = 0.23, P < 0.007], surface fibrin deposition [r(s) = 0.12, P < 0.01], fibrosis [r(s) = 0.31, P < 0.006], edema [r(s) = 0.17, P = 0.07], congestion [r(s) = 0.30, P < 0.005], and infiltration [r(s) = 0.46, P < 0.0001]). Fibrin and infiltration parameters were more severe in end-stage disease (P = 0.009 and P = 0.02, respectively). Synovitis may be present from the onset of OA and may be evaluated on MRI. MRI evaluation of synovitis could be used to classify OA patients in clinical trials and could help to identify those who could benefit from synovium-targeted therapy.

Evaluation of different techniques for percutaneous needle biopsy of synovial membrane in the dog

Four different needles were evaluated for percutaneous needle biopsy (PNB) of the synovial membrane (SM) in six different joints (carpus, tarsus, elbow, shoulder, stifle, hip) from eight dog cadavers. Needle biopsies were retrieved with the Parker-Pearson (PP), a novel modified Parker-Pearson (PPM), the Tru-Cut (TC), and the Franklin-Silverman (FS) needles. The success rate of PNB to produce biopsies, of adequate histological quality, was assessed for each needle type and joint. The joints were subsequently surgically opened and standard open SM biopsies were harvested from an adjacent area. The articular cartilage was inspected and graded for damage caused by PNB sampling. The length of the intact synovial lining cell layer (SLCL) and the histological quality of the needle biopsies were compared to the open surgical biopsies. Success rates for the different needles were 49.2% (PPM), 37.5% (PP), 26.7% (TC), and 1.7% (FS). The highest success rates were achieved in the carpus and stifle; the lowest success rate was found in the hip joint. The intact SLCL from the PNB samples was an average of 3.4 times shorter than that of the surgical biopsies. The proportion of the samples with adequate histological quality was 81% for open surgical biopsies and 62% for PNB samples. The FS was found to produce the highest incidence of articular cartilage damage. The application of PNB for SM sampling is a valuable alternative to the open surgical biopsy in dogs. Modifications of the Parker Pearson needle by Arassy and Schumacher and novel modifications introduced by the authors, offer encouraging results, and merit further clinical and research investigations.

Involvement of poly(ADP-ribose) polymerase 1 in ERBB2 expression in rheumatoid synovial cells

Hyperplasia of synovial lining cells is one of the main features of rheumatoid arthritis (RA). We previously reported that ERBB2 is highly expressed in RA synovial cells and that it plays an important role in their hyperproliferative growth. Recent findings have suggested that poly(ADP-ribose) polymerase-1 (PARP-1) is involved in the transactivation of NF-kappaB-dependent genes such as ERBB2. In the present study, we investigated the role of PARP-1 in ERBB2 transcription in RA synovial cells. The expression level of PARP-1 was significantly high in synovial cells derived from three patients with RA, compared with three patients with osteoarthritis (OA). Luciferase assays revealed that PARP-1 augments the transcription of the ERBB2 gene and that a region between -404 and -368 is responsible for this activation. A protein with an apparent molecular mass of 115 kDa was isolated mainly from nuclear extracts of RA synovial cells with an affinity matrix harboring a DNA fragment identical to the above region. Mass spectrometric analysis demonstrated this protein to be PARP-1. Southwestern blot analysis showed that PARP-1 binds to this region, but not to adjacent regions. PARP-1 associates directly with NF-kappaB, and a chromatin immunoprecipitation assay indicated that these proteins interact with this enhancer region in the ERBB2 gene. Treatment of RA synovial cells with PARP-1 small interfering RNA attenuated their ERBB2 expression, while an inhibitor of the polymerase activity of PARP-1 had no effect. PARP-1 DNA binding is not required for transcriptional activation. These findings suggest that PARP-1 is involved in the expression of ERBB2 in concert with NF-kappaB, which might be associated with the proliferation of RA synovial cells.

Various secretory phospholipase A2 enzymes are expressed in rheumatoid arthritis and augment prostaglandin production in cultured synovial cells

Although group IIA secretory phospholipase A2 (sPLA2-IIA) is known to be abundantly present in the joints of patients with rheumatoid arthritis (RA), expression of other sPLA2s in this disease has remained unknown. In this study, we examined the expression and localization of six sPLA2s (groups IIA, IID, IIE, IIF, V and X) in human RA. Immunohistochemistry of RA sections revealed that sPLA2-IIA was generally located in synovial lining and sublining cells and cartilage chondrocytes, sPLA2-IID in lymph follicles and capillary endothelium, sPLA2-IIE in vascular smooth muscle cells, and sPLA2-V in interstitial fibroblasts. Expression levels of these group II subfamily sPLA2s appeared to be higher in severe RA than in inactive RA. sPLA2-X was detected in synovial lining cells and interstitial fibers in both active and inactive RA sections. Expression of sPLA2-IIF was partially positive, yet its correlation with disease states was unclear. Expression of sPLA2 transcripts was also evident in cultured normal human synoviocytes, in which sPLA2-IIA and -V were induced by interleukin-1 and sPLA2-X was expressed constitutively. Adenovirus-mediated expression of sPLA2s in cultured synoviocytes resulted in increased prostaglandin E2 production at low ng x mL(-1) concentrations. Thus, multiple sPLA2s are expressed in human RA, in which they may play a role in the augmentation of arachidonate metabolism or exhibit other cell type-specific functions.

P290 Modulation of lubricin glycosylation and multimerization in synovial lining cells by TGF-b and leukemia inhibitory factor

P290 Modulation of lubricin glycosylation and multimerization in synovial lining cells by TGF-b and leukemia inhibitory factor

Expression of ADAM15 in rheumatoid synovium: up-regulation by vascular endothelial growth factor and possible implications for angiogenesis

ADAMs (a disintegrin and metalloproteinases) comprise a new gene family of metalloproteinases, and may play roles in cell-cell interaction, cell migration, signal transduction, shedding of membrane-anchored proteins and degradation of extracellular matrix. We screened the mRNA expression of 10 different ADAMs with a putative metalloproteinase motif in synovial tissues from patients with rheumatoid arthritis (RA) or osteoarthritis (OA). Reverse transcription PCR and real-time quantitative PCR analyses indicated that among the ADAMs, ADAM15 mRNA was more frequently expressed in the RA samples and its expression level was significantly 3.8-fold higher in RA than in OA (p < 0.01). In situ hybridization, immunohistochemistry and immunoblotting demonstrated that ADAM15 is expressed in active and precursor forms in the synovial lining cells, endothelial cells of blood vessels and macrophage-like cells in the sublining layer of RA synovium. There was a direct correlation between ADAM15 mRNA expression levels and vascular density in the synovial tissues (r = 0.907, p < 0.001; n = 20). ADAM15 was constitutively expressed in RA synovial fibroblasts and human umbilical vein endothelial cells (HUVECs), and the expression level was increased in HUVECs by treatment with vascular endothelial growth factor (VEGF)165. On the other hand, ADAM15 expression in RA synovial fibroblasts was enhanced with VEGF165 only if vascular endothelial growth factor receptor (VEGFR)-2 expression was induced by treatment with tumor necrosis factor-α, and the expression was blocked with SU1498, a specific inhibitor of VEGFR-2. These data demonstrate that ADAM15 is overexpressed in RA synovium and its expression is up-regulated by the action of VEGF165 through VEGFR-2, and suggest the possibility that ADAM15 is involved in angiogenesis in RA synovium.

若年者に発生した腰部脊柱管内滑膜嚢腫の一例

We report a case of intraspinal synovial cyst with lumber spondylolysis. A healthy 17-year-old man suffered low back pain in combination with dysethesia of the left lower limb. Radiographs and CT of the lumber spine demonstrated bilateral L5 spondylolysis. However, the facet joints were devoid of degenerative changes. MRI showed development of a cystic legion compressing the thecal sac at the L4-5 level. Laminectomy was performed and the cyst was resected. It lay posterior to the nerve roots and its base was related to the L4-5 facet joint. Histologically, a cyst wall of fibrous tissue with synovial lining cells and evidence of inflammation with granulation tissue were seen. The patient's symptoms disappeared completely after surgery. Hypermobility of the facet joint is apparently a predisposing factor of cyst formation.

Collagenases and aggrecanases: understanding the role of non-catalytic domains in cartilage matrix breakdown

Degradation of cartilage matrix, which mainly consists of collagen fibrils and aggrecan, seriously impairs the function of joints. The primary cause of this process is elevated proteolytic enzymes. Collagen fibrils are degraded by a group of matrix metalloproteinases (MMPs) including collagenases (MMP-1, MMP-8 and MMP-13), gelatinase A (MMP-2) and MT1-MMP (MMP-14). Aggrecan is degraded by MMPs and 'aggrecanases' belonging to the ADAMTS family. Inhibitors of these metalloproteinases are considered as potential therapeutic agents to protect cartilage degradation, and many active site-directed inhibitors with a zinc-chelating moiety have been developed. Unfortunately, those compounds have serious side effects possibly due to lack of selectivity [1]. In the hope of developing new types of inhibitors we have been investigating the mechanisms of action of collagenases and aggrecanases. Collagenases are unique as they can degrade triple helical interstitial collagens into three-quarter and one-quarter fragments, a crucial step for collagenolysis in the tissue. Typical collagenases consist of a catalytic domain and a hemopexin domain that are connected by a linker peptide. Both domains are essential for collagenolysis. The three-dimensional structure of a prototypic collagenase, collagenase 1 (MMP-1), however, indicated that the active site of the enzyme is too narrow to accommodate triple-helical collagen. We have recently shown that collagenase locally unwinds triple helical collagen before it hydrolyses the peptide bonds [2]. Based on a series of mutagenesis we predict that the collagen binding site is created by both the catalytic and the hemopexin domains. This potential collagen binding groove is partially blocked by the prodomain in the zymogen of MMP-1 (proMMP-1), explaining its inability to bind to collagen unless it is activated. Overall proMMP-1 has a 'closed' configuration in contrast to the 'open' configuration of the active MMP-1. The exact mechanism of how collagenase unwinds collagen is under investigation. Another key collagenase is MT1-MMP. It is highly expressed in rheumatoid synovial lining cells invading into the cartilage. Overexpression of recombinant MT1-MMP in COS7 cells in vitro degrade reconstituted type I collagen films. This reaction requires dimerisation of the enzyme on the cell surface through the hemopexin domains. When dimerisation of MT1-MMP is prevented by overexpression of the membrane-anchored MT1-MMP hemopexin domain, the collagenolytic activity was blocked, but not its general photolytic activity. Among 19 ADAMTSs in humans, six have been shown to have aggrecanase activity. Their non-catalytic domains include one disintegrin, one or more thrombospondin, one cysteine-rich and one spacer domain. Full-length ADAMTS-4 (aggrecanase 1) is active on aggrecan only, but when the C-terminal spacer domain is deleted it expresses broader proteolytic activity and digests fibronectin, fibromodulin, decorin and biglycan, as well as maintaining aggrecanase activity [3]. Further deletion of the cysteine-rich and thrombospondin domains greatly reduces both activities [3]. These studies suggest that activities of collagenases and aggrecanases will be attenuated by inhibitors or antibodies that interact directly with their non-catalytic ancillary domains (exosite/allosteric inhibitors). Such molecules will be attractive for therapy as they will be highly selective because they are based on the unique mechanism of each proteinase.

Immunocytochemical localization of MAPKAPK-2 and Hsp25 in the rat temporomandibular joint

One series of our research has shown an intense expression of immunoreaction for heat shock protein 25 (Hsp25) in various cellular elements in the rat temporomandibular joint (TMJ). This protein is the major substrate of mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK-2), which mediates an intracellular stress-activated signaling pathway to stimulate cytosolic actin reorganization under various stresses. The present study was undertaken to examine the localization of MAPKAPK-2 in the rat TMJ by immunocytochemical techniques. Furthermore, confocal microscopy with double staining was employed to demonstrate the colocalization of MAPKAPK-2 and Hsp25. Immunocytochemistry for MAPKAPK-2 showed an intense immunoreaction in the cytoplasm of the synovial lining cells, the endothelial cells, and the fibroblasts in the synovial membrane of the rat TMJ. Double immunostaining under a confocal microscope succeeded in demonstrating the colocalization of MAPKAPK-2 and Hsp25 immunoreactions in the cytoplasm of fibroblastic type B synoviocytes in the TMJ. On the other hand, the macrophage-like type A-cells expressed MAPKAPK-2 immunoreactions but lacked Hsp25 immunoreactivity. The cells in the articular disk and the chondrocytes in the maturative and hypertrophic layer of the mandibular cartilage also showed intense immunoreactions for MAPKAPK-2 and Hsp25. In addition to cytoplasmic localization, MAPKAPK-2 immunoreactions were found in the nucleus of some synovial lining cells, cells in the articular disk, and chondrocytes. Current observations imply the presence of the phosphorylation of Hsp25 via activated MAPKAPK-2 in the cytoplasm. MAPKAPK-2 and Hsp25 possibly participate in the induction of cytoskeletal changes to the various cellular elements in rat TMJ under normal conditions.

The relationship of VEGF and PGE2 expression to extracellular matrix remodelling of the tenosynovium in the carpal tunnel syndrome

Tenosynovial thickening within the confined space of the carpal tunnel is thought to be the cause of the carpal tunnel syndrome (CTS). However, little is known about the pathological mechanism of tenosynovial thickening. In this study, the role of prostaglandin E(2) (PGE(2)) and vascular endothelial growth factor (VEGF) (two representative molecules that can induce oedema by increasing vascular permeability) was analysed in CTS by using immunohistochemistry and enzyme-linked immunosorptive assay (ELISA). Expression of these molecules was compared with the patients' clinical histories and a temporary increase in production of these molecules was found in cells within the vessels and synovial lining during the intermediate phase of the syndrome when the histology of the tenosynovium changes from oedematous to fibrotic. Statistical analysis clearly demonstrated that there is a close correlation between the expression of PGE(2) and VEGF. Furthermore, immunohistochemical analysis with anti-proliferating cell nuclear antigen (PCNA) revealed that the area with distinct VEGF expression closely matched the area where endothelial cells, vascular smooth muscle cells, and synovial lining cells proliferate. In contrast, despite marked alteration in the extracellular matrix (ECM) component of the tenosynovium, the fibroblasts responsible for most ECM framework production do not proliferate during any phase of CTS. Histological analysis demonstrated that angiogenesis takes place only during the intermediate phase. Since clusters of capillaries and arterioles are often surrounded by type III collagen-rich, disorganized, degenerate connective tissue, which contains fewer fibroblasts than normal, angiogenesis appears to take place as a part of a regenerative reaction that results in fibrosis. These findings strongly indicate that both PGE(2) and VEGF are expressed in the tenosynovium in CTS during the intermediate phase and induce the histological changes seen in the tenosynovium.

Distinct contribution of IL-6, TNF-α, IL-1, and IL-10 to T cell–mediated spontaneous autoimmune arthritis in mice

Cytokines play key roles in spontaneous CD4+ T cell–mediated chronic autoimmune arthritis in SKG mice, a new model of rheumatoid arthritis. Genetic deficiency in IL-6 completely suppressed the development of arthritis in SKG mice, irrespective of the persistence of circulating rheumatoid factor. Either IL-1 or TNF-α deficiency retarded the onset of arthritis and substantially reduced its incidence and severity. IL-10 deficiency, on the other hand, exacerbated disease, whereas IL-4 or IFN-γ deficiency did not alter the disease course. Synovial fluid of arthritic SKG mice contained high amounts of IL-6, TNF-α, and IL-1, in accord with active transcription of these cytokine genes in the afflicted joints. Notably, immunohistochemistry revealed that distinct subsets of synovial cells produced different cytokines in the inflamed synovium: the superficial synovial lining cells mainly produced IL-1 and TNF-α, whereas scattered subsynovial cells produced IL-6. Thus, IL-6, IL-1, TNF-α, and IL-10 play distinct roles in the development of SKG arthritis; arthritogenic CD4+ T cells are not required to skew to either Th1 or Th2; and the appearance of rheumatoid factor is independent of joint inflammation. The results also indicate that targeting not only each cytokine but also each cell population secreting distinct cytokines could be an effective treatment of rheumatoid arthritis.

Oxidative stress-induced DNA damage in the synovial cells of the temporomandibular joint in the rat.

Synovial hyperplasia is a feature of degenerative temporomandibular joint (TMJ) disease. However, the mechanism by which hyperplasia progresses in the TMJ is unknown. Based on the hypothesis that the oxidative stress generated by mechanical loading causes degenerative changes in the TMJ synovium, we investigated the generation of the highly reactive species, peroxynitrite, and the occurrence of DNA damage in the synovium. After condylar hypermobility of rat TMJs, a marker of peroxynitrite, nitrotyrosine, was localized to the nuclei and cytoplasm of the synovial lining cells and fibroblasts in synovitis-induced TMJ. DNA single-strand breaks were found in the nuclei of the synovial cells only after enzyme treatment, whereas DNA double-strand breaks were not detected. These findings indicate that condylar hypermovement induces the proliferation of synovial cells, and suggest that oxidative stress leads to the progression of synovial hyperplasia via DNA damage of the synovial cells in TMJs after mechanical loading.

Elevation of activated protein C in synovial joints in rheumatoid arthritis and its correlation with matrix metalloproteinase 2.

To investigate the involvement of the anticoagulant serine protease activated protein C (APC) in tissue remodeling in rheumatoid arthritis (RA). PC/APC, matrix metalloproteinase 2 (MMP-2), and MMP-9 were detected in synovial fluid by Western blotting, and their antigen levels were quantified by enzyme-linked immunosorbent assay in patients with osteoarthritis (OA) or RA. Enzymatic activity of MMP-2 was assayed using a specific fluorogenic substrate. We developed an improved assay to measure APC activity in synovial fluid utilizing a chromogenic substrate following immunoprecipitation with a specific PC/APC antibody. PC/APC and MMP-2 were localized by immunohistochemistry in RA, OA, and normal synovial tissues. Synovial fluid analysis demonstrated that APC is present in both RA and OA synovial fluid, with APC activity being markedly higher in RA (mean +/- SEM 462 +/- 112 ng/ml versus 136 +/- 42 ng/ml; P < 0.02). A correlation (r(2) = 0.61) was found between APC and MMP-2 activity levels in RA patients, but not in OA patients. Immunohistochemical studies of synovial sections showed colocalization of APC and MMP-2 in endothelial and synovial lining cells. Additionally, APC and MMP-2 coimmunoprecipitated with an anti-PC/APC antibody. Our results show, for the first time, that APC and MMP-2 are coordinately up-regulated and tightly bound in RA synovial fluid and colocalized in synovia. Their association suggests that APC may modulate MMP-2 activity in RA.

Latent Epstein–Barr virus (EBV) infection and cytomegalovirus (CMV) infection in synovial tissue of autoimmune chronic arthritis determined by RNA- and DNA-in situ hybridization

In rheumatoid arthritis (RA) viral triggers, especially Epstein–Barr virus (EBV) and cytomegalovirus (CMV), have been suggested. By PCR analysis DNA of several viruses among which EBV, CMV, and parvovirus B19 (B19) has been detected in RA synovial fluid and synovial tissue. In 63 synovial tissues of 29 rheumatoid arthritis (RA), 6 psoriatic arthritis (PsA), 26 reactive arthritis/synovitis (rA/S), and two normal synovial cases, we recently could demonstrate a high percentage of replicative B19 infection within the synovial tissue, being significantly more frequent in autoimmune arthritis. To further investigate the influence of synovial virus infections in rheumatoid arthritis, we now analyzed the same sample of synovial tissues for CMV and EBV infections by DNA-in situ hybridization (CMV), EBER1/2-RNA-in situ hybridization (EBV), and immunohistochemistry. A significant latent EBV infection of synovial lining cells, synovial fibroblasts, and/or infiltrating lymphocytes was identified in 5/29 (17.2 %) RA, 1/6 (16.7%) PsA, and to a much lower degree in 1/26 (3.8%) rA/S specimens. CMV-DNA was detected in 31% of RA, 3/6 (50%) of PsA, and 11.5% of rA/S. Immunohistochemical analysis of CMV early antigen revealed replicative CMV activity in 20.7% of RA and 2/6 (33.3%) of PsA specimens but not in reactive arthritis synovia. Comparative analyis of the EBV-, CMV-, and published B19-data demonstrated that relevant synovial virus infections in general and furthermore double or multiple infections are far more common in autoimmune arthritis than in rA/S. A triple virus infection was found solely in RA in 10.3% of cases. The evidence of increased synovial persistence of EBV, CMV, or B19 either alone or even more as coinciding infections may further reinforce the notion of a primary role of these viruses in autoimmune arthritis.

Expression of microsomal prostaglandin E synthase 1 in rheumatoid arthritis synovium.

Microsomal prostaglandin E synthase 1 (mPGES-1) catalyzes the formation of PGE(2) from cyclooxygenase-derived PGH(2). Microsomal PGES-1 is induced by proinflammatory cytokines and is strongly linked to conditions that result in high PGE(2) biosynthesis. PGE(2) contributes to the pathogenesis of rheumatoid arthritis (RA), acting as a mediator of inflammation and promoting bone destruction. Induction of mPGES-1 in rheumatoid synoviocytes by proinflammatory cytokines has been demonstrated in vitro, indicating an important role in RA pathogenesis. Recent studies using mPGES-1-deficient mice demonstrated the importance of this gene in chronic inflammation. The aim of this study was to investigate the expression and localization of mPGES-1 in synovial biopsy specimens obtained from patients with RA. Synovial tissue samples from 24 patients with RA were obtained, and immunohistologic analysis was performed using polyclonal antibodies against mPGES-1. Double immunofluorescence staining was performed with antibodies to CD3, CD19, CD20, CD68, CD163, and prolyl 4-hydroxylase. Intracellular mPGES-1 staining was observed in synovial membranes from all of the RA patients studied. Specifically, strong expression of mPGES-1 was detected in synovial lining cells. In sublining mononuclear and fibroblast-like cells, the extent of mPGES-1 staining was less than that in the synovial lining cells. In some patients, positive staining was observed in endothelial cells. With the double immunofluorescence technique, mPGES-1 production was detected in synovial macrophages and fibroblasts, while mPGES-1 expression was not observed in lymphocytes. The demonstration of mPGES-1 expression in synovial tissues from patients with RA suggests a role for mPGES-1 in the RA disease process. Microsomal PGES-1 might be a potential new target for treatment strategies to control PGE(2) synthesis in patients with RA, without the systemic side effects associated with cyclooxygenase inhibitors.

Immunohistochemical localization of cyclooxygenase-1 and -2 in synovial tissues from patients with internal derangement or osteoarthritis of the temporomandibular joint

This study examined the immunohistochemical expression and localization of cyclooxygenase-1 and -2 (COX-1 and COX-2) in synovial tissues from patients with internal derangement (ID) or osteoarthritis (OA) of the temporomandibular joint (TMJ). Synovial tissues from patients with condylar fractures of the mandible were studied as control. Synovial tissues from 13 TMJs of 10 patients with ID or OA and from 5 TMJs of 4 patients with fractures were examined for COX-1 and COX-2 expression by immunohistochemical staining using two monoclonal antibodies. In addition, whether the COX-2 expression grade correlated with the synovitis score and clinical findings was assessed. COX-2 was expressed in the synovial lining, infiltrating mononuclear cells, fibroblast-like cells, and blood vessels, including CD31-positive endothelial cells, in the synovium of patients with ID or OA. Expression levels of COX-1 in synovial lining cells and endothelial cells were similar in the specimens obtained from the patients with ID or OA and those obtained from the controls. The expression of COX-2 positively correlated with arthroscopic findings of synovitis ( ρ=0.55, P=0.023) and with joint pain ( ρ=0.56, P=0.021). These results suggest that up-regulation of COX-2 in synovium may play a part in the pathogenesis of synovitis in patients with ID or OA of the TMJ.

Autocrine/paracrine role of the angiopoietin-1 and -2/Tie2 system in cell proliferation and chemotaxis of cultured fibroblastic synoviocytes in rheumatoid arthritis

Hypervascularity is a characteristic synovial feature of rheumatoid arthritis (RA). We previously reported that Tie1 and Tie2, endothelium-specific tyrosine kinase receptors essential for angiogenesis, are expressed not only by vascular cells, but also by a subpopulation of synovial lining and stromal cells in the inflamed RA synovium. The present study used immunohistochemistry and in situ hybridization to examine whether angiopoietin-1 and -2 (Ang1 and Ang2), ligands for Tie2, are also expressed in the RA synovium. Ang1 and Ang2 were expressed in all of 15 RA synovial samples, and their distribution pattern was similar to that of Tie2. Intense staining was noted in synovial lining and stromal cells, as well as in small vessels in areas of papillary projection and high cell density. Double immunohistochemistry revealed coexpression of Ang1, Ang2, and Tie2 in synovial components exhibiting proliferating cell nuclear antigen immunoreactivity. In addition, Ang1 and Ang2 were preferentially expressed in vimentin-positive fibroblastic cells. To address the functional role of Ang/Tie signaling in RA pathophysiology, we carried out [ 3H]thymidine incorporation and transwell chemotaxis assays using cultured fibroblastic synoviocytes obtained from 2 RA patients. Incubation with various concentrations of recombinant Ang1 or Ang2 did not alter DNA synthesis, but the ligands enhanced chemotactic migration of RA fibroblastic synoviocytes. Our results suggest that the autocrine/paracrine signaling of the Ang/Tie2 system is important for the up-regulated angiogenesis in the RA synovium, as well as for synoviocyte behavior, by regulating chemotactic cell movement.

Crucial role of synovial lining macrophages in the promotion of transforming growth factor beta-mediated osteophyte formation.

To investigate in vivo and in vitro whether macrophages have an intermediate role in transforming growth factor beta (TGFbeta)-induced osteophyte formation. In vivo, synovial lining macrophages were selectively depleted by injection of clodronate-laden liposomes 7 days prior to injection of 20 ng or 200 ng of TGFbeta into murine knee joints 3 times, on alternate days. Total knee joint sections were obtained on day 7 after the last injection and stained with Safranin O. Production of bone morphogenetic protein 2 (BMP-2) and BMP-4 was determined by immunolocalization. The interaction between murine macrophages and mesenchymal cells (precursors with chondrogenic potential) was studied in vitro using a Transwell system in which RAW macrophages were cocultured with C3H10T1/2 mesenchymal cells. Spheroid neocartilage formation was quantified microscopically after staining with May-Grünwald-Giemsa. Triple injections of 20 ng or 200 ng of TGFbeta into normal murine knee joints induced significant osteophyte formation at the lateral and medial sites of the patella and femur on day 7 after the last injection. Strikingly, removal of synovial lining macrophages prior to TGFbeta injection resulted in a drastic reduction of osteophyte formation (by 70% and 64% after injection of 20 ng and 200 ng of TGFbeta, respectively). Synovial lining cells produced BMP-2 and BMP-4 after TGFbeta stimulation, whereas BMP-2 and BMP-4 were absent in the synovial tissue after macrophage depletion. In vitro, clustering and spheroid formation of C3H10T1/2 was induced by TGFbeta concentrations of >1 ng/ml. However, in the Transwell system, in the presence of murine macrophages, 0.5 ng/ml of TGFbeta was very effective in generating large spheroids, suggestive of macrophage-derived (co)factors. In coculture supernatants, TGFbeta concentrations were not elevated in the presence of macrophages, indicating generation of other growth factors involved in spheroid formation. These findings indicate that macrophages are crucial intermediate factors in osteophyte formation induced by TGFbeta, probably by inducing other chondrogenic signals.