Detection and quantitation of stem cell factor (kit ligand) in the synovial fluid of patients with rheumatic disease

OBJECTIVE: To identify and quantitate stem cell factor (SCF; kit ligand) in the serum and synovial fluid (SF) of patients with rheumatoid arthritis (RA) and to compare these values with those measured in normal serum, RA serum, SF of patients with other rheumatic diseases, and conditioned medium from cultured synoviocytes. METHODS: SCF was measured in serum, SF, and conditioned synovial cell culture medium by a sensitive ELISA. Results were correlated with hematologic variables including white blood cell count, hemoglobin, platelet count, erythrocyte sedimentation rate, and rheumatoid factor. RESULTS: SCF levels in RA SF exceeded those measured in RA serum, osteoarthritis SF, and SF from patients with other inflammatory arthropathies. SCF was detectable in conditioned medium from cultured synoviocytes. CONCLUSION: High levels of SCF are present in RA serum and SF. Local production of SF may influence expansion of myeloid progenitor cells and mast cell function in RA.     

Regulation of bovine bone cell proliferation by fibroblast growth factor and transforming growth factor beta

We have tested the hypothesis that basic fibroblast growth factor (bFGF) and transforming growth factor beta (TGF beta) regulate the proliferation of osteoblast-like cells. Cells which migrated from central bone explants of fetal calf calvaria expressed markers characteristic of the osteoblast phenotype, including osteocalcin (bone Gla protein) secretion and increased cAMP production in response to treatment with PTH. Bone cells proliferated in response to bFGF in a dose- and time-dependent pattern (ED50 = 60 pg/ml media). bFGF increased both the rate of bone cell proliferation (1.7-fold above controls) and final cell density at confluence (3-fold above controls). Acidic FGF (aFGF) exerted comparable effects though with lesser potency (ED50 = 2 ng/ml). In addition to its mitogenic effect, bFGF increased the osteocalcin content of conditioned media, suggesting that bFGF also modulates the function of osteoblast-like cells. Although TGF beta did not stimulate bone cell proliferation, it potentiated the mitogenic effects of aFGF and bFGF. In the presence of bFGF (0.7 ng/ml) the response to TGF beta was dose-dependent (ED50 = 1.7 ng/ml), with maximal stimulation at 5 ng/ml. These results demonstrate that aFGF and bFGF are mitogenic for bone cells in vitro. Furthermore, TGF beta potentiates the effects of bFGF and aFGF on the proliferation of bone cells. Since these growth factors are present in bone tissue in vivo, these data support the proposal that FGF and TGF beta may participate in the regulation of bone formation.     

The effect of apoptosis signal-regulating kinase 1 gene transfer on rat collagen induced arthritis

OBJECTIVE: To examine the apoptosis-inducing effect of apoptosis signal-regulating kinase 1 (ASK1) gene transfer into synovial cells in vitro and in vivo. METHODS: An adenovirus vector was constructed so that a constitutively active form of ASK1 gene (ASK1DeltaN) was expressed in the presence of the Cre recombinase. The ASK1DeltaN and Cre adenovirus vectors were cotransduced into cultured synoviocytes derived from patients with rheumatoid arthritis (RA), and apoptosis was evaluated by TUNEL and Hoechst staining. Collagen induced arthritis (CIA) was induced in 8-week-old male DA rats, and 10 days later the 2 adenovirus vectors were coadministered into the ankle joints of the animals. As indicators of severity of arthritis, swelling of the ankle and articular index (AI) scores were evaluated, while histopathological observation of articular tissue was also performed. RESULTS: In the cultured human RA synoviocytes, overexpression of the ASK1DeltaN significantly reduced cell viability and induced apoptosis. In the CIA rats transduced with the ASK1DeltaN gene, arthritis was significantly promoted in terms of the swelling of the ankle joints and elevation of the AI scores. Histopathological observation also revealed that the constitutively active ASK1 induced massive infiltration of inflammatory cells into the synovial membrane as well as proliferation of synovial fibroblasts. Degeneration of the synovial membrane was not evident. CONCLUSION: Adenoviral transduction of ASK1DeltaN induced apoptosis in RA synoviocytes in vitro, but not in CIA synovium in vivo.     

Interaction of fibroblast growth factor (FGF) with megakaryocytopoiesis and demonstration of FGF receptor expression in megakaryocytes and megakaryocytic-like cells.

We have investigated the interaction of fibroblast growth factor (FGF) with megakaryocytopoiesis. Acidic FGF (aFGF) stimulated the proliferation of murine megakaryocytes and human erythroleukemia (HEL) cells in a concentration-dependent manner. The concentrations of aFGF required to elicit half-maximum and maximum effects were similar for HEL and megakaryocytic colony formation. The effect of aFGF was comparable to that of basic FGF (bFGF) in both cell types. The effect of both FGFs was found to be synergistic with interleukin-3 (IL-3), and was abrogated by a monoclonal anti-IL-6 antibody. A specific cell surface receptor complex of approximately 120 Kd was detected for FGF by crosslinking experiments on HEL cells and total bone marrow (BM) cells. Single-cell autoradiography of megakaryocytes in BM smears and BM cultures showed binding sites for 125I-aFGF. Northern blot analysis of messenger RNA (mRNA) from total BM and HEL cells showed a 4.4-kb mRNA specific for FGF receptors type 1 (flg) and type 2 (bek). This was confirmed by polymerase chain reaction, which also showed the presence of FGF receptor mRNA in megakaryocytic-like cells, normal megakaryocytes, and platelets. Together, these results indicate that FGF is involved in megakaryocytopoiesis and suggest that this interaction may be mediated via FGF receptor type 1 and type 2 located on the megakaryocytic lineage or on accessory cells responsible for the release of megakaryocytic growth-promoting activities.     

Cytokeratin Expression and Hyaluronic Acid Production in Cultures of Human Synovial Microvascular Endothelial Cells: Influence of Cytokines and Growth Factors

Objectives: To isolate and characterize human synovial endothelial cells and to determine the effects of cytokines and fibroblast growth factor on human synovial endothelial (HSE) cell hyaluronic acid production. Methods: Endothelial cells were isolated from primary cultures of human synovial cells by fluorescent activated cell sorting based on the incorporation of a fluorescent derivative of acetylated low-density lipoprotein (DiI-Ac-LDL). Identity of endothelial cells was confirmed by positive immunostaining for von Willebrand factor (vWf), cytokeratins, endoglin, and reactivity with the lectin ulex europeaus agglutinin (UEA). Hyaluronic acid production was measured by a radioligand-binding assay. Results: HSE cells were isolated and maintained in long-term culture. The identity of the cultured cells as endothelial was based on uniform uptake of a (DiI-Ac-LDL), immunoreactivity for vWf, and endoglin and the binding of the lectin UEA. In addition, small blood vessels in the synovium were stained selectively with anticytokeratin antibodies K4.62 (cytokeratin 19 specific) and K8.13 (reactive for cytokines 1, 5, 6, 7, 8, 10, 11, and 18). Isolated HSE cells also demonstrated immunoreactivity with these cytokeratin antibodies. The cytokeratins identified by the monoclonal antibody clone K8.13 demonstrated a diffuse, fibrillar staining pattern. The cytokeratin distribution revealed with monoclonal antibody K4.62 (cytokeratin 19) was also fibrillar; however, the majority of cells also demonstrated numerous punctate cytoplasmic vesicular structures. Treatment of HSE cells with interleukin-1α (IL-1α) or acidic fibroblast growth factor (aFGF), but not tumor necrosis factor (TNFα), dramatically reduced the vesicular structures staining with the K4.62 antibody. HSE cells produced hyaluronic acid (HA) at a constitutive rate of 200–800 ng/10⁵ cells/24 h, which could be upregulated when the cells were incubated with either IL-1α or aFGF. HA production was not significantly increased when HSE cells were incubated with TNFα, IL-4 or interferon-γ. Conclusions: Synovial microvascular endothelial cells produce and secrete HA and endothelial HA secretion is upregulated by IL-1 and aFGF. IL-1 and aFGF also reduce the number of vesicular-like structures immunoreactive with a monoclonal antibody to cytokeratin 19. These studies suggest that cytokine stimulation of local endothelial secretion and/or accumulation of HA may influence leukocyte adhesion to the synovial endothelium.     

Chondroprogenitor cells of synovial tissue

OBJECTIVE: To assess the chondrogenic potential of cells within the synovium. METHODS: Explants of synovium taken from various sites in the joint were embedded in agarose and cultured with transforming growth factor beta1 (TGFbeta1) to assess their chondrogenic potential. Isolated synovial cells were also tested for their chondrogenic potential by culturing them as aggregates in a chemically defined medium with TGFbeta1. Cartilage formation was determined with histologic staining and immunohistochemistry. The osteochondral potential of the isolated cells was also assessed after subcutaneous implantation of the cells, loaded into porous calcium phosphate ceramic cubes, in athymic mice. RESULTS: A total of 48 synovial explants were cultured in agarose with TGFbeta1. The formation of cartilage was observed in the outer region of 21 explants, and type II collagen was localized in that region by immunohistochemistry. A larger percentage of TGFbeta1+ explants from the inner synovium sites formed cartilage compared with those from the outer synovium sites. Chondrogenesis occurred in aggregates incubated with TGFbeta1 as early as day 7, and by day 14, all TGFbeta1+ aggregates demonstrated chondrogenesis. In contrast with the results of the in vitro aggregate assay for chondrogenesis, no formation of cartilage or bone was evident in any section containing synovial cell-loaded ceramic cubes that were harvested at either 3 or 6 weeks after implantation subcutaneously in athymic mice. CONCLUSION: Synovial explants and isolated synovial cells will undergo chondrogenesis when cultured in the presence of TGFbeta1. The data indicate a possible synovial origin for the chondrocytic cells found in rheumatoid pannus. Furthermore, these data are consistent with the clinical findings of synovial chondrogenesis leading to synovial chondromatosis.     

Effects of combinations of anti-rheumatic drugs on the production of vascular endothelial growth factor and basic fibroblast growth factor in cultured synoviocytes and patients with rheumatoid arthritis

OBJECTIVE: To examine whether different combinations of disease-modifying anti-rheumatic drugs (DMARDs), including bucillamine (BUC), gold sodium thiomalate (GST), methotrexate (MTX), salazosulphapyridine (SASP) and dexamethasone (DEX; a steroid), act by inhibiting the production of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in cultured synoviocytes, causing a decrease in their serum concentrations in patients with rheumatoid arthritis (RA). METHODS: The VEGF and bFGF concentrations in cultured synoviocytes and peripheral blood from patients with RA were measured by enzyme-linked immunosorbent assay and their serum concentrations were measured at two time points. RESULTS: BUC and GST inhibited VEGF production even when given alone, and a combination of BUC, GST and MTX with DEX also inhibited VEGF production. None of the DMARDs or DEX inhibited bFGF production when given alone, but a combination of SASP and GST inhibited the production of bFGF in cultured synoviocytes. Serum VEGF concentrations were significantly decreased 6 months after the commencement of medication compared with their concentrations before medication. CONCLUSION: Our results show that the effects of a combination of DEX with any two of BUC, GST, SASP and MTX on the production of VEGF and bFGF in cultured synoviocytes and on the serum concentrations of VEGF in patients with RA may be based on synergistic or additive effects of the drugs.     

Transforming growth factor beta1 gene polymorphism in rheumatoid arthritis

OBJECTIVE: Rheumatoid arthritis (RA) is a chronic inflammatory disease and synovial cells, antigen presenting cells, lymphocytes, and their cytokines might be associated with the disease. Transforming growth factor beta1 (TGFbeta1) has been reported to have important roles in unresolved inflammation, immune suppression, fibrosing processes, and angiogenesis. TGFbeta1 is highly expressed in joints in RA and is considered to be a regulator of anti-inflammation in RA. Polymorphisms of TGFbeta1 have been reported to be associated with the production of TGFbeta1 protein, and to increase the risk of acquiring several diseases. It was speculated that these polymorphisms might also be involved in RA, and therefore the TGFbeta1 codon 10 T869C polymorphism in a series of patients and controls was investigated. METHOD: A total of 155 patients with RA and 110 healthy subjects were studied. DNA was extracted from peripheral leucocytes and TGFbeta1 codon 10 T869C polymorphism was determined by polymerase chain reaction restriction fragment polymorphism. RESULTS: A significantly higher proportion of patients with RA with the T allele (CT type or TT type) was found compared with the CC type (p=0.039). CONCLUSION: The T allele, previously reported to be linked with production of TGFbeta1, may be associated with an increased risk of RA.     

Involvement of fibroblast growth factor-2 in joint destruction of rheumatoid arthritis patients.

OBJECTIVE: To investigate the effect of the synovial fluid from knee joints of rheumatoid arthritis (RA) patients with different severities of joint destruction on osteoclastogenesis and bone resorption. METHODS: Synovial fluid was harvested from the knee joints of 59 RA patients and 37 ostcoarthritis (OA) patients. RA patients with Larsen's knee grade 1-3 were classified as mild RA (n = 30) and those with grade 4 or 5 as severe RA (n = 29). Cytokine concentrations in synovial fluid were measured by ELISA. Osteoclastogenesis was measured by tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cell (MNC) formation in a co-culture of mouse osteoblastic cells and bone marrow cells, and bone resorption by 45Ca release from pre-labelled cultured neonatal mouse calvariae. RESULTS: The synovial fluid of severe RA patients significantly stimulated TRAP-positive MNC formation and 45Ca release compared to those of mild RA and OA patients. Among the bone-resorptive cytokines fibroblast growth factor-2 (FGF-2), tumour necrosis factor alpha (TNF-alpha), interleukin-1alpha (IL-1alpha), IL-6 and soluble IL-6 receptor (sIL-6R), only FGF-2 concentration in the synovial fluid was positively correlated to Larsen's grade, and severe RA patients showed significantly higher FGF-2 concentrations than mild RA patients. Osteoclastogenesis in a co-culture system which was stimulated by the synovial fluid of severe RA patients was significantly inhibited by a neutralizing antibody against FGF-2 and this inhibition was stronger than antibodies against other cytokines. CONCLUSION: The increase in endogenous FGF-2 levels in the synovial fluid of RA patients may play a role in the joint destruction by inducing osteoclastogenesis.