Involvement of PDCD5 in the regulation of apoptosis in fibroblast-like synoviocytes of rheumatoid arthritis

Apoptosis is reduced in the synovial tissue of patients with rheumatoid arthritis (RA), possibly due to decreased expression of pro-apoptotic genes. Programmed Cell Death 5 (PDCD5) has been recently identified as a protein that mediates apoptosis. Although PDCD5 is down-regulated in many human tumors, the role of PDCD5 in RA has not been investigated. Here we report that reduced levels of PDCD5 mRNA and protein are detected in RA synovial tissue (ST) and fibroblast-like synoviocytes (FLS) than in tissue and cells from patients with osteoarthritis (OA). We also report differences in the PDCD5 expression pattern in tissues from patients with these two types of arthritis. PDCD5 showed a scattered pattern in rheumatoid synovium compared with OA, in which the protein labeling was stronger in the synovial lining layer than in the sublining. We also observed increased expression and nuclear translocation of PDCD5 in RA patient-derived FLS undergoing apoptosis. Finally, overexpression of PDCD5 led to enhanced apoptosis and activation of caspase-3 in triptolide-treated FLS. We propose that PDCD5 may be involved in the pathogenesis of RA. These data also suggest that PDCD5 may serve as a therapeutic target to enhance sensitivity to antirheumatic drug-induced apoptosis in RA.     

Differential expression of the FAK family kinases in rheumatoid arthritis and osteoarthritis synovial tissues

The focal adhesion kinase (FAK) family kinases, including FAK and proline-rich kinase 2 (Pyk)2, are the predominant mediators of integrin αvβ3 signaling events that play an important role in cell adhesion, osteoclast pathology, and angiogenesis, all processes important in rheumatoid arthritis (RA). Using immunohistochemical and western blot analysis, we studied the distribution of phospho (p)FAK, pPyk2, pSrc, pPaxillin and pPLCγ in the synovial tissue (ST) from patients with RA, osteoarthritis (OA) and normal donors (NDs) as well as in RA ST fibroblasts and peripheral blood differentiated macrophages (PB MΦs) treated with tumor necrosis factor-α (TNFα) or interleukin-1β (IL1β). RA and OA STs showed a greater percentage of pFAK on lining cells and MΦs compared with ND ST. RA ST fibroblasts expressed pFAK at baseline, which increased with TNFα or IL1β stimulation. Pyk2 and Src were phosphorylated more on RA versus OA and ND lining cells and MΦs. pPyk2 was expressed on RA ST fibrobasts but not in MΦs at baseline, however it was upregulated upon TNFα or IL1β activation in both cell types. pSrc was expressed in RA ST fibroblasts and MΦs at baseline and was further increased by TNFα or IL1β stimulation. pPaxillin and pPLCγ were upregulated in RA versus OA and ND lining cells and sublining MΦs. Activation of the FAK family signaling cascade on RA and OA lining cells may be responsible for cell adhesion and migration into the diseased STs. Therapies targeting this novel signaling pathway may be beneficial in RA.     

MDM4 overexpression contributes to synoviocyte proliferation in patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease with features of inflammatory cell infiltration, synovial cell invasive proliferation, and ultimately, irreversible joint destruction. It has been reported that the p53 pathway is involved in RA pathogenesis. MDM4/MDMX is a major negative regulator of p53. To determine whether MDM4 contributes to RA pathogenesis, MDM4 mRNA and protein expression were assessed in fibroblast-like synoviocytes (FLS) by real-time PCR, western blotting, and in synovial tissues by immunohistochemistry. Furthermore, MDM4 was knocked down and overexpressed by lentivirus-mediated expression, and the proliferative capacity of FLS was determined by MTS assay. We found that cultured FLS from RA and osteoarthritis (OA) patients exhibited higher levels of MDM4 mRNA and protein expression than those from trauma controls. MDM4 protein was highly expressed in the synovial lining and sublining cells from both types of arthritis. Finally, MDM4 knockdown inhibited the proliferation of RA FLS by enhancing functional p53 levels while MDM4 overexpression promoted the growth of RA FLS by inhibiting p53 effects. Taken together, our results suggest that the abundant expression of MDM4 in FLS may contribute to the hyperplasia phenotype of RA synovial tissues.     

Decrease in expression of bone morphogenetic proteins 4 and 5 in synovial tissue of patients with osteoarthritis and rheumatoid arthritis

Bone morphogenetic proteins (BMPs) have been identified as important morphogens with pleiotropic functions in regulating the development, homeostasis and repair of various tissues. The aim of this study was to characterize the expression of BMPs in synovial tissues under normal and arthritic conditions. Synovial tissue from normal donors (ND) and from patients with osteoarthritis (OA) and rheumatoid arthritis (RA) were analyzed for BMP expression by using microarray hybridization. Differential expression of BMP-4 and BMP-5 was validated by semiquantitative RT-PCR, in situ hybridization and immunohistochemistry. Activity of arthritis was determined by routine parameters for systemic inflammation, by histological scoring of synovitis and by semiquantitative RT-PCR of IL-1β, TNF-α, stromelysin and collagenase I in synovial tissue. Expression of BMP-4 and BMP-5 mRNA was found to be significantly decreased in synovial tissue of patients with RA in comparison with ND by microarray analysis (p < 0.0083 and p < 0.0091). Validation by PCR confirmed these data in RA (p < 0.002) and also revealed a significant decrease in BMP-4 and BMP-5 expression in OA compared with ND (p < 0.015). Furthermore, histomorphological distribution of both morphogens as determined by in situ hybridization and immunohistochemistry showed a dominance in the lining layer of normal tissues, whereas chronically inflamed tissue from patients with RA revealed BMP expression mainly scattered across deeper layers. In OA, these changes were less pronounced with variable distribution of BMPs in the lining and sublining layer. BMP-4 and BMP-5 are expressed in normal synovial tissue and were found decreased in OA and RA. This may suggest a role of distinct BMPs in joint homeostasis that is disturbed in inflammatory and degenerative joint diseases. In comparison with previous reports, these data underline the complex impact of these factors on homeostasis and remodeling in joint physiology and pathology.     

Detection of high levels of heparin binding growth factor-1 (acidic fibroblast growth factor) in inflammatory arthritic joints

The synovium from patients with rheumatoid arthritis (RA) and LEW/N rats with streptococcal cell wall (SCW) arthritis, an experimental model resembling RA, is characterized by massive proliferation of synovial connective tissues and invasive destruction of periarticular bone and cartilage. Since heparin binding growth factor (HBGF)-1, the precursor of acidic fibroblast growth factor (FGF), is a potent angiogenic polypeptide and mitogen for mesenchymal cells, we sought evidence that it was involved in the synovial pathology of RA and SCW arthritis. HBGF-1 mRNA was detected in RA synovium using the polymerase chain reaction technique, and its product was immunolocalized intracellularly in both RA and osteoarthritis (OA) synovium. HBGF-1 staining was more extensive and intense in synovium of RA patients than OA and correlated with the extent and intensity of synovial mononuclear cell infiltration. HBGF-1 staining also correlated with c-Fos protein staining. In SCW arthritis, HBGF-1 immunostaining was noted in bone marrow, bone, cartilage, synovium, ligamentous and tendinous structures, as well as various dermal structures and developed early in both T-cell competent and incompetent rats. Persistent high level immunostaining of HBGF-1 was only noted in T-cell competent rats like the disease process in general. These observations implicate HBGF-1 in a multitude of biological functions in inflammatory joint diseases.     

Characterization and functional consequences of underexpression of clusterin in rheumatoid arthritis

We previously compared by microarray analysis gene expression in rheumatoid arthritis (RA) and osteoarthritis (OA) tissues. Among the set of genes identified as a molecular signature of RA, clusterin (clu) was one of the most differentially expressed. In the present study we sought to assess the expression and the role of CLU (mRNA and protein) in the affected joints and in cultured fibroblast-like synoviocytes (FLS) and to determine its functional role. Quantitative RT-PCR, Northern blot, in situ hybridization, immunohistochemistry, and Western blot were used to specify and quantify the expression of CLU in ex vivo synovial tissue. In synovial tissue, the protein was predominantly expressed by synoviocytes and it was detected in synovial fluids. Both full-length and spliced isoform CLU mRNA levels of expression were lower in RA tissues compared with OA and healthy synovium. In synovium and in cultured FLS, the overexpression of CLU concerned all protein isoforms in OA whereas in RA, the intracellular forms of the protein were barely detectable. Transgenic overexpression of CLU in RA FLS promoted apoptosis within 24 h. We observed that CLU knockdown with small interfering RNA promoted IL-6 and IL-8 production. CLU interacted with phosphorylated IkappaBalpha. Differential expression of CLU by OA and RA FLS appeared to be an intrinsic property of the cells. Expression of intracellular isoforms of CLU is differentially regulated between OA and RA. We propose that in RA joints, high levels of extracellular CLU and low expression of intracellular CLU may enhance NF-kappaB activation and survival of the synoviocytes.     

Expression and regulation of aggrecanase in arthritis: the role of TGF-beta.

Aggrecanases are key matrix-degrading enzymes that act by cleaving aggrecan at the Glu(373)-Ala(374) site. While these fragments have been detected in osteoarthritis (OA) and rheumatoid arthritis (RA) cartilage and synovial fluid, no information is available on the regulation or expression of the two key aggrecanases (aggrecanase-1 and aggrecanase-2) in synovial tissue (ST) or fibroblast-like synoviocytes (FLS). The aggrecanase-1 gene was constitutively expressed by both RA and OA FLS. Real-time PCR demonstrated that TGF-beta significantly increased aggrecanase-1 gene expression in FLS. Aggrecanase-1 induction peaked after 24 h of TGF-beta stimulation. The expression of aggrecanase-1 mRNA was significantly greater in RA ST than in OA or nonarthritis ST. Aggrecanase-2 mRNA and protein were constitutively produced by nonarthritis, OA, and RA FLS but were not increased by IL-1, TNF-alpha, or TGF-beta. Furthermore, OA, RA, and nonarthritis ST contained similar amounts of immunoreactive aggrecanase-2. The major form of the aggrecanase-2 enzyme was 70 kDa in nonarthritis ST, whereas a processed 53-kDa form was abundant in RA ST. Therefore, aggrecanase-1 and -2 are differentially regulated in FLS. Both are constitutively expressed, but aggrecanase-1 is induced by cytokines, especially TGF-beta. In contrast, aggrecanase-2 protein may be regulated by a post-translational mechanism in OA and RA ST. Synovial and FLS production of aggrecanase can contribute to cartilage degradation in RA and OA.     

Enzymatic activity and distribution of phospholipase A2 in human cartilage

Extracellular phospholipase A2 (PLA2) with proinflammatory activity has recently been discovered in synovial fluids in inflammatory arthritides. In the search for the sources of synovial fluid PLA2, human synovium and articular cartilage were found to contain large quantities of the enzyme. In rheumatoid arthritis (RA), PLA2 activity in synovium, superficial and deep layers of articular cartilage was 20 +/- 14 (SEM), 168 +/- 62 and 533 +/- 176 nmol/min/mg protein respectively. Corresponding values in osteoarthritis (OA) were 49 +/- 11, 569 +/- 109 and 1709 +/- 243 nmol/min/mg protein, all significantly higher (p less than .01) than in RA. Nasal septal cartilage contained much less PLA2, 19 +/- 5.6. PLA2 in human articular and nasal cartilage has sn-2 specificity, a neutral pH optimum and absolute calcium dependence. High PLA2 concentration in articular cartilage may imply that, at least in part, cartilage is the source of PLA2 in the joint space. Since RA cartilage and synovium have less PLA2 activity than the corresponding OA tissues, additional sources of PLA2 in RA synovial fluids are implicated.     

Alpha 4/beta 1 integrin (VLA-4) ligands in arthritis. Vascular cell adhesion molecule-1 expression in synovium and on fibroblast-like synoviocytes.

Expression of vascular cell adhesion molecule-1 (VCAM-1) in synovial tissue was determined using the immunoperoxidase technique. Normal, rheumatoid arthritis (RA), and osteoarthritis (OA) synovia bound VCAM-1 antibodies in the intimal lining as well as blood vessels. The amount of VCAM-1 was significantly greater in the synovial lining of RA and OA tissues compared with normal synovium (p less than 0.002). There was also a trend toward greater levels of VCAM-1 staining in blood vessels of arthritic tissue (RA greater than OA greater than normal). Because VCAM-1 staining was especially intense in the synovial lining, VCAM-1 expression and regulation was studied on cultured fibroblast-like synoviocytes (FLS) derived from this region. Both VCAM-1 and intercellular adhesion molecule 1 were constitutively expressed on FLS. VCAM-1 expression was further increased by exposure to IL-1 beta, TNF-alpha, IL-4, and IFN-gamma. These cytokines (except for IL-4) also induced intercellular adhesion molecule 1 expression on FLS. ELAM was not detected on resting or cytokine-stimulated FLS. The specificity of VCAM-1 for FLS was demonstrated by the fact that only trace amounts were detected on normal and RA dermal fibroblasts. Cytokines induced intercellular adhesion molecule 1 display on dermal fibroblasts but had minimal effect on VCAM-1 expression. Finally, in adherence assays, Jurkat cell binding to resting FLS monolayers was inhibited by antibody to alpha 4/beta 1 integrin (VLA-4), CS-1 peptide from alternatively spliced fibronectin (which is another VLA-4 ligand), and, to a lesser extent, anti-VCAM-1 antibody. After cytokine stimulation of FLS, Jurkat-binding significantly increased, and this increase was blocked by anti-VCAM-1 antibody. Therefore, both CS-1 and VCAM-1 participate in VLA-4-mediated adherence to resting FLS in vitro, and VCAM-1 is responsible for the increase in Jurkat binding mediated by cytokines.     

Fibroblast activation protein is expressed by rheumatoid myofibroblast-like synoviocytes

Fibroblast activation protein (FAP), as described so far, is a type II cell surface serine protease expressed by fibroblastic cells in areas of active tissue remodelling such as tumour stroma or healing wounds. We investigated the expression of FAP by fibroblast-like synoviocytes (FLSs) and compared the synovial expression pattern in rheumatoid arthritis (RA) and osteoarthritis (OA) patients. Synovial tissue from diseased joints of 20 patients, 10 patients with refractory RA and 10 patients with end-stage OA, was collected during routine surgery. As a result, FLSs from intensively inflamed synovial tissues of refractory RA expressed FAP at high density. Moreover, FAP expression was co-localised with matrix metalloproteinases (MMP-1 and MMP-13) and CD44 splice variants v3 and v7/8 known to play a major role in the concert of extracellular matrix degradation. The pattern of signals appeared to constitute a characteristic feature of FLSs involved in rheumatoid arthritic joint-destructive processes. These FAP-expressing FLSs with a phenotype of smooth muscle actin-positive myofibroblasts were located in the lining layer of the synovium and differ distinctly from Thy-1-expressing and non-proliferating fibroblasts of the articular matrix. The intensity of FAP-specific staining in synovial tissue from patients with RA was found to be different when compared with end-stage OA. Because expression of FAP by RA FLSs has not been described before, the findings of this study highlight a novel element in cartilage and bone destruction of arthritic joints. Moreover, the specific expression pattern qualifies FAP as a therapeutic target for inhibiting the destructive potential of fibroblast-like synovial cells.     

Mcl-1 is essential for the survival of synovial fibroblasts in rheumatoid arthritis

Mcl-1 is a Bcl-2-family, antiapoptotic molecule that is critical for the survival of T and B lymphocytes and macrophages; however, its role in nonhemopoietic cells remains to be fully elucidated. The current study focuses on the role of Mcl-1 in rheumatoid arthritis (RA). Mcl-1 was strongly expressed in the synovial lining and was increased in the sublining fibroblasts of patients with RA, compared with control synovial tissue. The expression of Mcl-1 in sublining fibroblasts correlated with the degree of inflammation and TNF-alpha, and IL-1beta treatment of cultured synovial fibroblasts resulted in the increased expression of Mcl-1 at the mRNA and protein levels. Mcl-1 was critical for the survival of RA synovial fibroblasts, because the forced reduction of Mcl-1 using a Mcl-1 antisense-expressing adenoviral vector induced apoptotic cell death, which was mediated through Bax, Bak, and Bim. These observations document a critical role for Mcl-1 in protecting against apoptosis in RA and suggest that Mc1-1 is a potential therapeutic target in this disease.     

Intimal lining layer macrophages but not synovial sublining macrophages display an IL-10 polarized-like phenotype in chronic synovitis

Introduction

Synovial tissue macrophages play a key role in chronic inflammatory arthritis, but the contribution of different macrophage subsets in this process remains largely unknown. The main in vitro polarized macrophage subsets are classically (M1) and alternatively (M2) activated macrophages, the latter comprising interleukin (IL)-4 and IL-10 polarized cells. Here, we aimed to evaluate the polarization status of synovial macrophages in spondyloarthritis (SpA) and rheumatoid arthritis (RA).

Methods

Expression of polarization markers on synovial macrophages, peripheral blood monocytes, and in vitro polarized monocyte-derived macrophages from SpA versus RA patients was assessed by immunohistochemistry and flow cytometry, respectively. The polarization status of the intimal lining layer and the synovial sublining macrophages was assessed by double immunofluorescence staining.

Results

The expression of the IL-10 polarization marker cluster of differentiation 163 (CD163) was increased in SpA compared with RA intimal lining layer, but no differences were found in other M1 and M2 markers between the diseases. Furthermore, no significant phenotypic differences in monocytes and in vitro polarized monocyte-derived macrophages were seen between SpA, RA, and healthy controls, indicating that the differential CD163 expression does not reflect a preferential M2 polarization in SpA. More detailed analysis of intimal lining layer macrophages revealed a strong co-expression of the IL-10 polarization markers CD163 and cluster of differentiation 32 (CD32) but not any of the other markers in both SpA and RA. In contrast, synovial sublining macrophages had a more heterogeneous phenotype, with a majority of cells co-expressing M1 and M2 markers.

Conclusions

The intimal lining layer but not synovial sublining macrophages display an IL-10 polarized-like phenotype, with increased CD163 expression in SpA versus RA synovitis. These differences in the distribution of the polarized macrophage subset may contribute to the outcome of chronic synovitis.