Department of Internal Medicine, Division of Rheumatology, 1 Department of Experimental Pathology and Oncology, University of Florence, Florence, 2 Institute of Clinical Medicine, Hematology and Clinical Immunology, University of Ancona, Ancona, 3 Department of Internal Medicine and Public Health, University of L'Aquila, L'Aquila and 4 Medical Direction, Laboratori Guidotti S.p.a., Pisa, Italy.
Correspondence to: M. Matucci Cerinic, Department of Internal Medicine, Division of Rheumatology, University of Florence, Viale Pieraccini 18, 50139, Firenze, Italy. E-mail: cerinic{at}unifi.it
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods. uPA, uPAR and PAI-1 levels were studied by ELISA, RT-PCR (uPAR) and zymography (uPA) in synoviocytes from four RA patients and four healthy controls. Chemoinvasion was assessed by the Boyden chamber invasion assay, using Matrigel as the invasion substrate. Proliferation was evaluated by cell counting. Both invasion and proliferation were measured upon treatment with deflazacort 5 µM with or without parallel stimulation with uPA 500 ng/ml or in the presence of monoclonal anti-uPA and anti-uPAR antibodies.
Results. Invasion and proliferation of RA synoviocytes require a proper functional balance of the fibrinolytic system. Both deflazacort and monoclonal antibodies against uPA and uPAR reduced expression and activity of the system, thus inhibiting invasion and proliferation. In RA synoviocytes, deflazacort induced higher PAI-1 and lower uPA and uPAR levels, as well as a decrease in uPA enzymatic activity. The levels of uPAR mRNA were concomitantly reduced, as was uPA-induced chemoinvasion. All these effects were also shown in controls, though to a lesser extent.
Conclusions. Deflazacort might control RA synovial proliferation and invasion by differential modulation of single members of the fibrinolytic system.
KEY WORDS: Deflazacort, Rheumatoid Arthritis, Fibrinolysis, uPA, Synoviocytes, Steroids
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
RA synoviocytes display proliferation and invasion properties similar to transformed cells, resulting in formation of a hypertrophic synovial pannus which also erodes cartilage and subchondral bone [1]. The growth/invasive phenotype is characteristic of transformed cells and is now recognized as being mainly dependent on aberrant expression of cell-associated protease systems. Other conditions are paralleled by aberrant proteolysis, such tumour- or inflammation-associated angiogenesis [2, 3] and breakdown of the articular cartilage in osteoarthritis [4].
Two classes of membrane-associated proteases, the fibrinolytic and the matrix metalloproteinase (MMPs) systems, cooperate in extracellular proteolysis.
In the fibrinolytic system, the urokinase-type plasminogen activator (uPA) interacts with its membrane receptor (uPAR) and activates the single-chain proenzyme plasminogen (PLG) to the two-chain broad-spectrum serine proteinase plasmin (PL), which degrades extracellular matrix (ECM) both directly or indirectly, through activation of secreted pro-MMPs.
Activation of PLG occurs on receptors for PLG/PL present on several cell lines [5]. Complex and not yet fully understood crosstalk between the fibrinolytic and MMP systems is an issue of active research [6]. In RA, synoviocytes exhibit a hyperinvasive phenotype, which has been mainly related to an overall up-regulation of the fibrinolytic system. These data refer to observations performed on tissue specimens [7, 8], where the effects of the intra-articular microenvironment play a pivotal role. Data from our laboratory, obtained in isolated RA synoviocytes, while confirming up-regulation of uPAR and PAI-1 [9, 10], indicate down-regulation of uPA expression and production [10]. In the in vivo setting, synoviocytes are likely to pick up uPA produced under stimulation of inflammatory cytokines by synoviocytes themselves or by the inflammatory cells that are abundant in the RA joint, as indicated by detection of plasminogen activator activity in cultures of human monocytes, chondrocytes and synoviocytes, where their production can be regulated by a variety of cytokines produced in diseased joints [8, 11].
Upon isolation, RA synoviocytes recover their constitutive environment-independent phenotype, which has been suggested to resemble the premature phenotype of primordial synoviocytes [12]. Whatever the case, in the RA joint the abundance of uPA triggers a multienzyme cascade leading to ECM destruction and cell progression in the underlying tissues. Serine proteases are involved in cartilage degradation both by direct activation of latent MMPs, which have a main role in cartilage breakdown, and by inducing a potent pro-angiogenic stimulus that influences the synovial pannus growth.
uPA/uPAR interaction induces plasmin-dependent events, such as chemoinvasion, and also plasmin-independent events, such as chemotaxis and chemokinesis [13], proliferation [14, 15], differentiation and autocrine secretion of uPA [16]. Our group has shown that, in healthy synoviocytes, uPA/uPAR interaction determines dose-dependent chemotaxis, chemoinvasion and proliferation [17].
The final evolution of RA is erosion of bone and cartilage and joint ankylosis. The aim of using disease-modifying anti-rheumatic drugs (DMARDs) is to interrupt this trend, particularly in the early phases of the disease, in order to prevent bone erosions.
The hypothesis that steroids may have an effect on joint destruction has been re-evaluated. Several papers have addressed this problem and have supported the theory that steroids, in early RA, may significantly reduce the rate of radiological progression [18, 19]. Indeed, the withdrawal of these drugs led to a significant deterioration of the disease with an overshoot of radiographic progression [20].
Deflazacort (DFZ) is an oxazoline derivative of prednisolone with anti-inflammatory and immunosuppressive activity. In general, DFZ appears less active than other steroids on the parameters associated with the development of corticosteroid-induced osteoporosis [21].
DFZ is commonly used in the treatment of RA, for its efficacy and safety [21]. However, the effects of DFZ on synoviocytes are not clear and little is known about the relationships between the fibrinolytic system and DFZ in healthy and RA synoviocytes.
Our aim was to study the effects of DFZ on RA synoviocytes to understand if the drug may modulate the membrane-bound fibrinolytic system and if it may interfere with uPA-dependent cellular proliferation and invasion, in order to evaluate the potential future use of this drug in the control of RA-associated bone erosion and cartilage breakdown.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Synovial cell cultures
Synovia was removed from knee joints, cut and subjected to a mild proteolytic treatment (0.05% trypsin, 0.5 mM EDTA in phosphate-buffered saline, for 10 min at 37°C with gentle shaking). Trypsin was neutralized with fetal calf serum (FCS) (Celbio, Milan, Italy) and cells were plated in culture dishes with RPMI 1640 (Cambrex BioScience, Milan, Italy) supplemented with 10% FCS, 2 mM glutamine (Cambrex) and penicillinstreptomycin (Cambrex). The cell monolayers were used within the 7th passage in culture. The cells were considered type B fibroblast-like synovial cells if negative on staining with anti-CD69, anti-CD14, anti-CD11b and anti-CD11c (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and positive by staining for uridine diphosphoglucose dehydrogenase (UDPGD), and if they had a spindle-shaped, fibroblast-like morphology.
DFZ (Guidotti, Pisa, Italy) was dissolved in acetic acid 100 mmol and further diluted with culture medium. Samples were analysed both in basal conditions and after treatment with DFZ (48 h). In preliminary experiments we tested the effects of different doses of DFZ, in order to identify the maximal dose without lethal effects. Thus, the concentrations of 3 µM and 5 µM DFZ were chosen for further experiments and used in this study. Such concentrations are in agreement with those reported for other corticosteroids in vitro [22].
Proliferation assay
Cell growth was quantified in subconfluent cell monolayers. Synoviocytes were seeded in 24 multi-well plates (15 000 cells/well) with 10% FCS in RPMI 1640. After 48 h incubation, cells were washed three times with serum-free medium and incubated in 0.2% FCS medium for an additional 48 h. Then, cells were incubated for 48 h in 10% FCS medium, or in 0.2% FCS medium, or in 0.2% FCS with uPA (Serono, Rome, Italy) 500 ng/ml with/without uPA or uPAR antagonists. These were anti-human uPA monoclonal antibody (mAb) 5B4 and anti-uPAR mAb 3936 (mAb 3936) (American Diagnostica, Montreal, Canada), which were used at 1.5 µg/ml. The mAb 5B4 and 3936 both sterically impeded uPA/uPAR interaction. In preliminary experiments we tested the effects of different doses of uPA, in order to identify which dose could be more efficacious in inducing synoviocyte proliferation. Thus, the concentration of 500 ng/ml was chosen.
Each experiment was performed in triplicate. At the end of incubation cells were counted.
Samples were analysed both in basal conditions and after treatment with DFZ 3 µM and 5 µM (48 h).
Migration assays
The Boyden chamber procedure was used to evaluate cell migration [17]. The method is based on the passage of cells across porous filters against a concentration gradient of the migration effector. A 48-well micro-chemotaxis chamber (Neuroprobe, Gaithersburg, MD, USA) was used. The two wells were separated by a polyvinyl pyrrolidine-free polycarbonate filter, 8 µm pore size (Neuroprobe). To evaluate chemoinvasion, the filter was coated with Matrigel (50 µg/filter) (Becton Dickinson, Bedford, MA, USA). Test solutions were dissolved in serum-free medium and placed in the lower wells. Cell suspension (50 µl, 12 500 cells) was added to the upper and/or lower well. uPA 100 ng/ml and/or DFZ (3 and 5 µM) was added to the lower well. In the experiment with neutralizing antibodies, the anti-uPA mAb 5B4 (1.5 µg/ml) was placed in the lower wells, while the anti-uPAR mAb 3936 (1.5 µg/ml) was incubated with the cell suspension. Irrelevant mouse IgGs were used at the same concentration in both the upper and lower well, to verify the specificity of the effect. The chamber was incubated at 37°C for 5 h, and the filter was then removed and fixed with methanol. Non-migrating cells on the upper surface of the filter were removed with a cotton swab. Cells were stained with Diff-Quick (Mertz-Dade, Dade International, Milan, Italy) and counted by a light microscope (40x) in 10 random fields for each well. Mobilization was measured by the number of cells moving across the Matrigel and the filter pores and spread on the lower surface of the filter. Each experiment was performed in triplicate. Mean values of migrated cells for each experimental point were calculated.
Samples were analysed both in basal conditions and after treatment with DFZ (48 h).
DFZ 3 µM and 5 µM were used for migration assays.
Analysis of uPA, uPAR and PAI-1 levels
Samples were analysed for uPAR, uPA and PAI-1 by commercially available enzyme-linked immunoassay kits (Imubind; American Diagnostica) according to the manufacturer's instructions. Briefly, synoviocytes were seeded in six multi-well plates (25 000 cells/well) with 10% FCS in RPMI 1640. After 48 h of incubation, cells were washed three times with serum-free medium and incubated in 0.2% FCS medium for an additional 48 h. Then, cells were treated with DFZ 3 and 5 µM for 48 h. At the end of incubation, cells were detached, counted and lysed, as suggested by the manufacturer. The lysates were replaced in their original well and incubated for 1 h at 4°C to allow exhaustive extraction of undetached material. Cell extracts were centrifuged and stored at 80°C till uPAR analysis. Culture media were collected, centrifuged and stored at 80°C till uPA and PAI-1 determination. The results were correlated to the standard curve, within the range of linearity. Each sample was evaluated in triplicate and with two different dilutions.
The sensitivity levels were: 10 pg of uPA/ml of sample; 0.1 ng of uPAR/ml of sample; 1 ng of PAI-1 /ml of sample.
Analysis of uPA enzymatic activity
uPA enzymatic activity was evaluated by zymography. Culture medium samples were collected as described above and concentrated by centrifugation at 8000 r.p.m. for 30 min in Centricon tubes (Amicon, Beverly, MA, USA) with 30 kDa molecular weight cut-off pores. The samples were subjected to sodium dodecyl sulphate (SDS)polyacrylamide slab gel electrophoresis (10%) under non-reducing conditions and migrated proteins were transferred onto 0.45 µm pore-size nitrocellulose filter (Bio-Rad Laboratories, Richmond, CA, USA) in 0.04 M phosphate buffer (pH 6.5), with a current of 0.4 A during a 2 h run. The nitrocellulose filter was removed and placed on an indicating layer containing casein and plasminogen. After overnight incubation at 37°C, when clear bands of lysis were visible in the cloudy casein background, corresponding to the position of plasminogen activators in the polyacrylamide gel, zymograms were dried and stained with Ponceau S solution (Sigma). uPA activity was measured by densitometric scanning of the zymograms against standard uPA.
Samples were analysed both in basal conditions and after treatment with DFZ 3 and 5 µM (48 h).
uPAR gene mRNA quantitation by reverse transcriptase-polymerase chain reaction
The mRNA level of the uPAR gene was determined by an internal standard-based quantitative reverse transcriptasepolymerase chain reaction (RT-PCR) assay. Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was chosen as the internal standard gene because of its expression at a constant per-cell level.
Total RNA was extracted according to the method of Chomczynski and Sacchi [23] and reverse-transcribed to cDNA. uPAR and GAPDH gene segments were separately amplified using serial dilutions of cDNA.
cDNA synthesis, PCR and quantification of amplified products were performed as previously described [24].
The uPAR gene primers were: sense 5'-GGT CAC CCG CCG CTG-3', antisense 5'-CCA CTG CGG TAC TGG ACA-3'. The GAPDH gene primers were: sense 5'-CCA CCC ATG GCA AAT TCC ATG GCA-3', antisense 5'- TCT AGA CGG CAG GTC AGG TCC ACC-3'.
Values of integrated densities were plotted as a function of the increasing amount of amplified reverse transcriptase products. At least three values of each gene PCR product, falling within the linear range of amplification, were normalized to the starting cDNA volumes and referred to the values of PCR products relative to the GAPDH mRNA used as internal standard.
Samples were analysed both in basal conditions and after treatment with DFZ 3 and 5 µM (48 h).
Statistics
The non-parametric MannWhitney test for independent samples was used to compare results from healthy and RA synoviocytes for the levels of uPA, uPAR and PAI-1. The results were expressed as mean ± S.D.
Zymography was evaluated by densitometric comparison between lysis areas produced by uPA. The proliferation induced by uPA and DFZ was evaluated by the two-tailed t-test for independent samples and by analysis of variance (ANOVA) with Bonferroni correction.
Migration was expressed as the mean ± S.E.M. of the percentage of the healthy basal response, which was considered as 100%.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
These results showed that uPA exerted a proliferative effect both on healthy and RA synoviocytes, and indicated 500 ng/ml uPA as the minimal dose providing the maximal proliferative effect. This dose was used in the following experiments on uPA-dependent proliferation.
Effects of DFZ on synoviocyte proliferation
DFZ inhibited in a dose-dependent manner cell proliferation in both H and RA synoviocytes (P<0.001 for any dose in both cell lines), reaching a maximum at 5 µM (Fig. 2A).
|
These results indicated that DFZ exerted an antiproliferative effect on both H and RA synoviocytes and that DFZ was able to induce this effect even at 3 µM.
Effects of DFZ on uPA-dependent proliferation
In both normal and RA synoviocytes, DFZ significantly reduced uPA-dependent and 10% FCS-challenged proliferation (P<0.001 in both case), until reaching the basal values (P>0.05) (Fig. 2B).
As shown in Fig. 2B, RA synoviocytes were as prone as normal synoviocytes to spontaneous (0.2% FCS) proliferation and to proliferation induced by 10% FCS, as well as 500 ng/ml uPA (P>0.05 in all cases).
In particular, serum-dependent proliferation (10% FCS) did not significantly differ from uPA-dependent proliferation in both H and in RA lines.
The proliferative effect elicited by 500 ng/ml uPA in 0.2% FCS was significantly reduced by the mAb antagonists of uPA and uPAR in H and RA cells (P<0.001) (Fig. 2B). This indicates that uPA/uPAR interaction is required for the proliferative effect of uPA on normal and RA synoviocytes.
DFZ blocked the effect of uPA by reducing the specific interactions between uPA and uPAR: in fact DFZ reduction of uPA-challenged proliferation was not different from the reduction of uPA proliferation induced by 5B4 and 3936 (Fig. 2B) and was not increased by cotreatment with them, indicating a common target for DFZ and anti-uPA/uPAR antibodies (data not shown). Alternatively, our data do not exclude the possibility that DFZ, as well as antibody-dependent antagonization of uPA/uPAR interaction, could affect ligandreceptor downstream signalling events, which are known to be critical for uPAR-dependent activity.
uPA, uPAR and PAI-1 levels in normal and RA synoviocytes
uPA released in the culture medium by RA synoviocytes was significantly lower than uPA produced by normal cells (3.57 ± 0.20 vs 11.19 ± 0.33 ng/106 cells, P<0.05) (Fig. 3A).
|
On the contrary, significantly higher levels of uPAR were measured in cell lysates of RA synoviocytes than of healthy ones (21.86 ± 1.63 vs 9.44 ± 0.31 ng/106 cells, P<0.05) (Fig. 3B).
Moreover, RA synoviocytes released into the culture medium significantly higher amounts of PAI-1 than healthy ones (7.62 ± 0.30 vs 3.67 ± 0.46 µg/106 cells, P<0.05) (Fig. 3C).
Effects of DFZ on uPA, uPAR and PAI-1 levels in normal and RA synoviocytes
DFZ significantly reduced uPA levels in H and RA synoviocytes (P<0.0001) (Fig. 3A). DFZ reduced uPA levels dose-dependently: DFZ 5 µM was more efficacious than DFZ 3 µM in H (P<0.05) and RA synoviocytes (P<0.01). DFZ-dependent reduction of uPA levels at 3 and 5 µM was significantly higher (P<0.05) in healthy than in RA synoviocytes.
Zymographic assays performed on culture medium from cells treated with DFZ 3 and 5 µM confirmed the data obtained with uPA. DFZ reduced uPA enzymatic activity in a dose-dependent manner, both in H and in RA synoviocytes (data not shown).
DFZ significantly (P<0.0001) reduced uPAR levels in cell lysates from H and RA synoviocytes (Fig. 3B). DFZ 5 µM was more efficacious than DFZ 3 µM in reducing uPAR levels in RA synoviocytes (P<0.01), but not in healthy ones (P>0.05). The effects of DFZ 3 and 5 µM were significantly higher (P<0.05) in RA than in healthy synoviocytes.
DFZ significantly increased (P<0.0001) PAI-1 levels in healthy and RA synoviocytes (Fig. 3C). This was true both for DFZ 3 and DFZ 5 µM (healthy synoviocytes, P<0.01 and P<0.001, respectively; RA, P<0.001). DFZ 5 µM was more efficacious than DFZ 3 µM in increasing PAI-1 levels, both in healthy and in RA synoviocytes (P<0.001).
Effects of DFZ on uPAR gene expression
To confirm the data obtained on the protein, we performed RT-PCR analysis of the specific uPAR mRNA, which provided the data shown in Fig. 4. The expression of the uPAR gene was normalized with respect to GAPDH constitutive gene expression. Gene expression resembled protein expression: healthy lines showed less uPAR than RA lines and treatment with DFZ decreased uPAR expression dose-dependently both in H and RA synoviocytes.
|
uPA-dependent synoviocyte chemoinvasion
uPA-dependent chemoinvasion was dose-dependent (Fig. 5A), with a maximal effect at 100 ng/ml for both healthy and RA synoviocytes. Basal migration was more pronounced (more than 40%) in RA than in healthy synoviocytes. This difference was maintained at each concentration of uPA in the lower well of the migration chamber. The increase in invasion observed after 5 h of incubation with 100 ng/ml uPA was counteracted by the incubation of invasive cells with mAb antagonists of uPA and uPAR, indicating that uPA/uPAR interaction is required for the invasive effect of uPA on normal and pathological synoviocytes (Fig. 5B).
|
DFZ induced a proportional decrease in invasion in synoviocytes from both sources, but such a decrease did not differ significantly between healthy and RA cells at any DFZ dose.
DFZ significantly reduced uPA-induced chemoinvasion in healthy and RA synoviocytes. DFZ 5 µM reduced chemoinvasion to basal values.
When DFZ was incubated in the upper chamber with the cells, this drug caused similar effects on chemoinvasion. In fact, DFZ was able to inhibit migration induced by uPA in a dose-dependent way, with maximal action at the concentration of 5 µM, and this decrease in mobility was proportionally similar in H and RA synoviocytes (data not shown).
DFZ blocked uPA-dependent chemioinvasion reducing uPAR expression and, therefore, the specific interactions between uPA and uPAR. DFZ reduction of uPA-dependent migration was similar to the inhibition caused by the antagonist antibodies (Fig. 5B).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Despite increasing evidence of the significance of corticosteroids in RA, their aetiopathological role and potential long-term effect on RA progression remain unclear. The link between the mechanisms of action of glucocorticoids and the fibrinolytic system is still to be elucidated in RA synoviocytes.
In RA, the formation and invasiveness of synovial pannus, supported by angiogenesis, is linked to serine proteinases, mainly uPA [7], present in high quantity in the RA joint and produced by monocytes, chondrocytes and synoviocytes themselves under the effects of a variety of cytokines [8, 11].
The uPA/uPAR/PAI-1 system is an organizer of cell-ECM contacts and covers the full range of activities required to promote cell invasion and to disrupt cell attachment sites. We have recently shown that RA synoviocytes display a fibrinolytic machinery (uPA, uPAR and PAI-1) addressed towards an invasive pattern [10]. DFZ inhibits RA synoviocyte proliferation dose-dependently and blocks the proliferation induced by uPA acting on the specific interactions between uPA and uPAR.
Cellular migration, linked to cellular adhesiveness, is an important process for the invasion of articular cavity and extra-articular tissues, typical of RA. Our data show that DFZ inhibits migration of RA synoviocytes dose-dependently, thus potentially contributing to the modulation of the growth of synovial pannus.
uPAR regulates pericellular proteolysis and cell surface adhesion receptors, fundamental events in the first steps of cell invasion and angiogenesis. The overexpression of uPAR in RA may depend on the need to activate the fibrinolytic pathway in order to degrade and invade ECM, as well as to promote interaction between uPAR and vitronectin, which provides the adhesive grip necessary for cell locomotion, events required in all the invasive pathologies [27]. DFZ lowers protein levels and mRNA levels of uPAR, exerting inhibition of the membrane-bound fibrinolytic system, which is required to stop also the proteolytic activities of MMPs.
DFZ further reduces uPA activity and levels in RA synoviocytes, thus potentially reducing both the invasive properties and the angiogenic potential of these cells, which may consist in providing uPA to the endothelial cells of the synovial membrane.
The increased expression of PAI-1 in RA synoviocytes is in agreement with previous data [7, 9, 10, 17]. In RA, elevated PAI-1 could act as an ECM-stabilizing molecule by blocking the extracellular proteolysis that provides cells with a substrate favouring cell movement. At the same time, PAI-1 promotes cell detachment [6, 27]. DFZ increases PAI-1, blocking uPA-dependent ECM degradation and reducing cell movement. The whole in vitro scenario is that DFZ reduces uPAR and uPA levels and increases PAI-1 levels. This activity may significantly contribute to reduce the invasiveness of RA synoviocytes, although the clinical relevance of these findings requires further study.
Since the seminal observation by Hamilton et al. [28], indicating that synoviocytes are a significant source of granulocyte-macrophage colony stimulating factor (GM-CSF) and granulocyte colony stimulating factor (G-CSF), a large number of effector chemokines and cytokines have been identified as being produced by synoviocytes, including those that promote bone resorption by stimulating osteoclastogenesis [29]. Given the broad biological effects of steroids, the determination of associations between uPA, uPAR and PAI-1 and concentrations of relevant autocrine mediators of synovial inflammation will be necessary to interpret our data on DFZ in a broader context.
In conclusion, DFZ modulates in vitro the components and the functionality of the membrane-bound fibrinolytic system in RA synoviocytes. The effect of DFZ in reducing formation of synovial pannus and the radiological progression of RA warrants further study.
|
The authors have declared no conflicts of interest.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|