**2.1 Experimental methods**

118 Rheumatoid Arthritis – Etiology, Consequences and Co-Morbidities

Fig. 1. Stratification of synovitis in relationship to fibrin deposits in a representative

Low-magnification microphotographs of a representative rheumatoid synovial tissue with an overlying fibrin deposit. Left column shows staining of the different tissue regions with haematoxylin-eosin. Middle column shows cell distribution with anti actin antibodies. On the right side, immune-localization of macrophages with anti CD68 antibodies is shown.

**2. Fibrin contributes to the production of MMP-1 and MMP-3 by rheumatoid** 

In order to test whether deposition of fibrin might trigger the production of MMPs, we investigated the presence of fibrin in synovial tissues from patients with rheumatoid arthritis. We observed a similar pattern of distribution to those of MMP-1 and -3. These findings led us to explore in vitro whether fibrin could activate the production of MMPs by rheumatoid synovial fibroblasts. In these studies, which we next describe, we introduced an

rheumatoid synovial tissue

**synovial fibroblasts** 

### **2.1.1 Obtention and handling of samples**

Synovial tissues were obtained during joint replacement surgery from 8 patients with rheumatoid arthritis according to the American College of Rheumatology criteria (Arnett et al., 1988). For histologic studies, the synovial membrane was immediately fixed in formaldehyde, dehydrated in ethanol and embedded in paraffin. When tissues included bone edges, they were decalcified by a 48 hour incubation in formic acid. For in vitro studies, cells were isolated by disruption with 1.5 mg/ml dispase II at 37ºC for 1 hour in agitation, and cultured in 10% fetal calf serum (FCS) enriched Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine, 50 UI/ml penicillin-streptomycin, 0.2% amphotericin B, and 10 mM HEPES.

#### **2.1.2 Immune-detection techniques**

We studied the distribution of fibrin, MMP-1 and MMP-3 with double labelling immunedetection methods. Tissues were rehydrated, blocked with 6% bovine albumin and 3% serum of second antibody hosts, and incubated with the specific antibodies at 10 g/ml overnight, at 4ºC. Secondary antibodies were applied for 1 hour, at room temperature. As control, tissues were incubated with an isotype IgG from the species of primary antibodies. Development of fibrinogen immune-reactivity was done with peroxidase and Histogreen, using nuclear fast red for counterstaining. Diaminobenzidine was applied after a byotinilated secondary antibody to develop MMP-3, and counterstaining was done with hematoxylin. An alkaline phosphatase labelled antibody was employed to detect MMP-1, and nitroblue tetrazolium with 5-bromo 4-chloro 3-indolyl phosphate was used as substrate for development, plus nuclear fast red as counterstaining. Inhibition of endogenous peroxidase was done with 1% H2O2 methanol. Alkaline phosphatase activity was blocked with 5 mM levamisole.

#### **2.1.3 Fibrin-cell cultures**

Between passages 4th and 7th cells were grown to confluence at 37ºC in 5% CO2, starved from serum during 48 hours and exposed to in situ clotted fibrin. Chilled fibrinogen was mixed in 0.5% foetal calf serum enriched DMEM at 1 mg/ml on ice, and 0.75 UI/ml thrombin was added. The mixture was immediately spread on top of the cell monolayers and the cultures were transferred to the incubator to allow formation of fibrin clots.

#### **2.1.4 Gene expression studies**

Four different cell cultures were employed. Cells were incubated with fibrin or medium alone for 12 hours. At the end of the incubation period, the clots and supernatants were removed, cells were washed and total RNA was isolated. Following retrotranscription, gene expression studies were done with quantitative PCR (qPCR) using cDNA as templates and

Innate Mechanisms of Synovitis – Fibrin Deposits Contribute to Invasion 121

3' (fwd), 5'-cacggttggagggaaaccta-3' (rev), FAM5'-agctggatacccaagaggcatccacac-3'TAMRA (probe); MMP 9: 5'-ggccactactgtgcctttgag-3' (fwd), 5'-gatggcgtcgaagatgttcac-3' (rev), FAM5' ttgcaggcatcgtccaccgg-3'TAMRA (probe); MMP 13: 5'-tcctacaaatctcgcgggaat-3' (fwd), gcatttctcggagcctctca-3' (rev), FAM5'-catggagcttgctgcattctccttcag-3'TAMRA (probe); MMP 14: 5'-tggaggagacacccactttga-3' (fwd), 5'-gccaccaggaagatgtcatttc-3' (rev), FAM5' cctgacagtccaaggctcggcaga-3'TAMRA (probe); urokinase: 5'-tgtcagcagccccactactac-3' (fwd),

**2.2.1 Fibrin and MMPs co-localized in the synovial tissues from patients with** 

We studied the distribution of fibrin(ogen) in synovial tissues from 8 patients with rheumatoid arthritis. Three of them included areas of invasion into cartilage and bone. The binding was strong and abundant in all samples, showing either an amorphous or a reticular pattern, as has been described (Andersen & Gormsen, 1970; Clemmensen et al., 1983). Fibrin predominated in the vicinity of the lining layer. Solid-looking deposits were mostly acellular, while more organized material was found in interstitial areas, with cells inside also capturing the antibody. Fibrin immune-reactivity was strong at areas of interface

Next, we studied the distribution of MMP-1 and MMP-3 in serial cuts of the same tissues, alone and in combination with fibrin, using double-staining methods. Immune-reactivity to both MMPs was high in the rheumatoid synovial tissues. Interestingly, MMP-1 predominated inside cells, and MMP-3 was mostly secreted. The binding of both antibodies

In double-staining studies, interstitial immune-reactivity to both MMP-1 and MMP-3 was associated with fibrin deposits. Furthermore, both proteases co-localized with fibrin at the invasive fronts. Fibroblast-like cells in fibrin-rich areas depicted a strong immune-reactivity to MMP-1 and MMP-3. These findings indicated that fibrin-rich areas were at the same time

**2.2.2 Gene expression of proteases in rheumatoid synovial fibroblasts exposed to** 

fibrin on the production of proteases by rheumatoid synovial fibroblasts.

In view of these observations, we carried out in vitro studies to look into a possible effect of

Thus, we studied five members of the MMP family that are prominent effectors of cartilage and bone destruction in rheumatoid arthritis. This revealed that exposure to in situ clotted fibrin resulted in the up-regulation of the gene expression of MMP-1, MMP-3, and MMP-9 to a variable extent in all cell cultures (Figure 3). The mRNA levels of MMP-1 were 26-fold increased (range: 1,5 to 71; p < 0.07), and those of MMP-3 were 27-fold increased (range: 2 to 126; p < 0.07). MMP-9 gene expression was increased 7-fold the presence of fibrin (range: 3 to 39; p < 0.07), while no differences were found in the gene expression of MMP-13 and MMP-14. Additionally, we studied urokinase gene expression, since it is the constitutive fibrindegrading molecule, and a potential mediator of tissue injury in rheumatoid synovial tissues. In our conditions, fibrin did not elicit any changes in urokinase mRNA levels

5'-cacagcattttggtggtgac-3' (rev).

**2.2 Results** 

**rheumatoid arthritis** 

with bone and cartilage.

was high at erosive fronts.

**fibrin clots** 

compared to baseline.

active sites of protease production.

TaqMan primer-probe reagents (MMPs) or SYBR Green techniques (urokinase). Results were analysed following the Ct method, using the expression of ribosomal 18s as house keeping gene and an untreated culture as reference.

### **2.1.5 Immunoblot techniques**

Confluent cells were incubated with or without fibrin and protein levels of MMPs were measured at several time points. The protein levels of MMP-1 were determined in cells lysates as obtained with Laemmli's solution. Supernatants and fibrin were mixed with 150 mM NaCl, 10 mM Tris, pH 7.2, 0.1% sodium dodecyl sulphate, 1% Triton X-100, 1% sodium deoxycholate and 5 mM ethylenediamine tetraacetic actid, with 1 g/ml aprotinin, 1 g/ml leupeptin, 1 mM Na3VO4, and 1 mM phenylmethylsulfonylfluoride, at 4ºC and homogenised. One hundred l of the homogenates were incubated with the anti MMP-3 antibody for 1 hour at 4ºC. Then, protein A/G plus-agarose immunoprecipitation reagent (20 l) was added, and samples were incubated overnight on a rocking platform at 4ºC. Finally, samples were washed and centrifuged to pellet beads with the complexed MMP-3, supernatants were discarded and precipitates were mixed in loading buffer. Protein extracts and immuno-precipitates were resolved in 10% SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose filters. The filters were then blocked with 5% skimmed milk and 0.03% Tween and blotted overnight with the specific antibodies. Horseradish peroxidase-labelled secondary antibodies were applied for 1 hour, at room temperature, and the binding was developed with enhanced chemiluminescence. Results were expressed as relative increase with regard to baseline. Betatubulin levels were used as control of cell protein content, and the Coomassie blue staining method was used in supernatants.

#### **2.1.6 Statistics**

Data are expressed as median (range); comparison between conditions was done with the non-parametric Wilcoxon rank test, using SPSS software.

#### **2.1.7 Reagents and probes**

Mouse anti human MMP-1 and goat anti human MMP-3 (R&D systems, Basel, Switzerland), goat anti human Fibrinogen (Abcam, Cambridge, UK), donkey anti goat-HRP, goat anti mouse-HRP, rabbit anti goat biotinylated antibodies, 1:500 in IHC, 1:10000 in WB (Jackson ImmunoResearch, LaRoche, Switzerland), goat anti mouse-alkaline phosphatase antibodies 1:40, Histogreen, nuclear fast red, DAB, peroxidase ABComplex (Dako, Zug, Switzerland), NBT-BCIP (Roche), Protein A/G Plus Agarose Immunoprecipitation Reagent (Santa Cruz Biotechnology Inc., CA), ECL chemoluminiscence (Amersham Pharmacia Biotech, Little Chalfont, UK) Fetal calf serum, DMEM, penicillin, streptomycin, HEPES, fungizone (Life Technologies, Basel, Switzerland), dispase (Roche, Reinach, Switzerland), fibrinogen (American Diagnostica inc., Stamford, CT), mouse anti human tubulin, thrombin (Sigma Aldrich, Buchs, Switzerland) MiniRNeasy spin column purification kit, RNase-free DNase set (Qiagen, Basel, Switzerland), SYBR green master mix, ABI Prism 7500 Sequence Detector (Applied Biosystems, Rotkreuz, Switzerland). Primer pairs and probes: MMP 1: 5' tgtggaccatgccattgaga-3' (fwd), 5'-tctgcttgaccctcagagacc-3' (rev), FAM5' ccaactctggagtaatgtcacacctctgacattcacc-3'TAMRA (probe); MMP 3: 5'-gggccatcagaggaaatgag3' (fwd), 5'-cacggttggagggaaaccta-3' (rev), FAM5'-agctggatacccaagaggcatccacac-3'TAMRA (probe); MMP 9: 5'-ggccactactgtgcctttgag-3' (fwd), 5'-gatggcgtcgaagatgttcac-3' (rev), FAM5' ttgcaggcatcgtccaccgg-3'TAMRA (probe); MMP 13: 5'-tcctacaaatctcgcgggaat-3' (fwd), gcatttctcggagcctctca-3' (rev), FAM5'-catggagcttgctgcattctccttcag-3'TAMRA (probe); MMP 14: 5'-tggaggagacacccactttga-3' (fwd), 5'-gccaccaggaagatgtcatttc-3' (rev), FAM5' cctgacagtccaaggctcggcaga-3'TAMRA (probe); urokinase: 5'-tgtcagcagccccactactac-3' (fwd), 5'-cacagcattttggtggtgac-3' (rev).

#### **2.2 Results**

120 Rheumatoid Arthritis – Etiology, Consequences and Co-Morbidities

TaqMan primer-probe reagents (MMPs) or SYBR Green techniques (urokinase). Results were analysed following the Ct method, using the expression of ribosomal 18s as house

Confluent cells were incubated with or without fibrin and protein levels of MMPs were measured at several time points. The protein levels of MMP-1 were determined in cells lysates as obtained with Laemmli's solution. Supernatants and fibrin were mixed with 150 mM NaCl, 10 mM Tris, pH 7.2, 0.1% sodium dodecyl sulphate, 1% Triton X-100, 1% sodium deoxycholate and 5 mM ethylenediamine tetraacetic actid, with 1 g/ml aprotinin, 1 g/ml leupeptin, 1 mM Na3VO4, and 1 mM phenylmethylsulfonylfluoride, at 4ºC and homogenised. One hundred l of the homogenates were incubated with the anti MMP-3 antibody for 1 hour at 4ºC. Then, protein A/G plus-agarose immunoprecipitation reagent (20 l) was added, and samples were incubated overnight on a rocking platform at 4ºC. Finally, samples were washed and centrifuged to pellet beads with the complexed MMP-3, supernatants were discarded and precipitates were mixed in loading buffer. Protein extracts and immuno-precipitates were resolved in 10% SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose filters. The filters were then blocked with 5% skimmed milk and 0.03% Tween and blotted overnight with the specific antibodies. Horseradish peroxidase-labelled secondary antibodies were applied for 1 hour, at room temperature, and the binding was developed with enhanced chemiluminescence. Results were expressed as relative increase with regard to baseline. Betatubulin levels were used as control of cell protein content, and the Coomassie blue

Data are expressed as median (range); comparison between conditions was done with the

Mouse anti human MMP-1 and goat anti human MMP-3 (R&D systems, Basel, Switzerland), goat anti human Fibrinogen (Abcam, Cambridge, UK), donkey anti goat-HRP, goat anti mouse-HRP, rabbit anti goat biotinylated antibodies, 1:500 in IHC, 1:10000 in WB (Jackson ImmunoResearch, LaRoche, Switzerland), goat anti mouse-alkaline phosphatase antibodies 1:40, Histogreen, nuclear fast red, DAB, peroxidase ABComplex (Dako, Zug, Switzerland), NBT-BCIP (Roche), Protein A/G Plus Agarose Immunoprecipitation Reagent (Santa Cruz Biotechnology Inc., CA), ECL chemoluminiscence (Amersham Pharmacia Biotech, Little Chalfont, UK) Fetal calf serum, DMEM, penicillin, streptomycin, HEPES, fungizone (Life Technologies, Basel, Switzerland), dispase (Roche, Reinach, Switzerland), fibrinogen (American Diagnostica inc., Stamford, CT), mouse anti human tubulin, thrombin (Sigma Aldrich, Buchs, Switzerland) MiniRNeasy spin column purification kit, RNase-free DNase set (Qiagen, Basel, Switzerland), SYBR green master mix, ABI Prism 7500 Sequence Detector (Applied Biosystems, Rotkreuz, Switzerland). Primer pairs and probes: MMP 1: 5' tgtggaccatgccattgaga-3' (fwd), 5'-tctgcttgaccctcagagacc-3' (rev), FAM5' ccaactctggagtaatgtcacacctctgacattcacc-3'TAMRA (probe); MMP 3: 5'-gggccatcagaggaaatgag-

keeping gene and an untreated culture as reference.

staining method was used in supernatants.

non-parametric Wilcoxon rank test, using SPSS software.

**2.1.6 Statistics**

**2.1.7 Reagents and probes** 

**2.1.5 Immunoblot techniques** 

#### **2.2.1 Fibrin and MMPs co-localized in the synovial tissues from patients with rheumatoid arthritis**

We studied the distribution of fibrin(ogen) in synovial tissues from 8 patients with rheumatoid arthritis. Three of them included areas of invasion into cartilage and bone. The binding was strong and abundant in all samples, showing either an amorphous or a reticular pattern, as has been described (Andersen & Gormsen, 1970; Clemmensen et al., 1983). Fibrin predominated in the vicinity of the lining layer. Solid-looking deposits were mostly acellular, while more organized material was found in interstitial areas, with cells inside also capturing the antibody. Fibrin immune-reactivity was strong at areas of interface with bone and cartilage.

Next, we studied the distribution of MMP-1 and MMP-3 in serial cuts of the same tissues, alone and in combination with fibrin, using double-staining methods. Immune-reactivity to both MMPs was high in the rheumatoid synovial tissues. Interestingly, MMP-1 predominated inside cells, and MMP-3 was mostly secreted. The binding of both antibodies was high at erosive fronts.

In double-staining studies, interstitial immune-reactivity to both MMP-1 and MMP-3 was associated with fibrin deposits. Furthermore, both proteases co-localized with fibrin at the invasive fronts. Fibroblast-like cells in fibrin-rich areas depicted a strong immune-reactivity to MMP-1 and MMP-3. These findings indicated that fibrin-rich areas were at the same time active sites of protease production.

#### **2.2.2 Gene expression of proteases in rheumatoid synovial fibroblasts exposed to fibrin clots**

In view of these observations, we carried out in vitro studies to look into a possible effect of fibrin on the production of proteases by rheumatoid synovial fibroblasts.

Thus, we studied five members of the MMP family that are prominent effectors of cartilage and bone destruction in rheumatoid arthritis. This revealed that exposure to in situ clotted fibrin resulted in the up-regulation of the gene expression of MMP-1, MMP-3, and MMP-9 to a variable extent in all cell cultures (Figure 3). The mRNA levels of MMP-1 were 26-fold increased (range: 1,5 to 71; p < 0.07), and those of MMP-3 were 27-fold increased (range: 2 to 126; p < 0.07). MMP-9 gene expression was increased 7-fold the presence of fibrin (range: 3 to 39; p < 0.07), while no differences were found in the gene expression of MMP-13 and MMP-14. Additionally, we studied urokinase gene expression, since it is the constitutive fibrindegrading molecule, and a potential mediator of tissue injury in rheumatoid synovial tissues. In our conditions, fibrin did not elicit any changes in urokinase mRNA levels compared to baseline.

Innate Mechanisms of Synovitis – Fibrin Deposits Contribute to Invasion 123

was well known. A novel finding is that all three followed a similar pattern of distribution. We could also confirm their abundance at CPJs, although we cannot draw conclusions as

Our studies suggest that fibrin triggers the transcription of several MMPs and the production of MMP-1. However, the study of MMPs is complex. These molecules are secreted in the form of zymogens, and need in situ activation through the cleavage of an Nterminus peptide. This post-translational modification probably constitutes a major checkpoint for the regulation of MMPs. Additionally, to find out what the global matrix turn-over could be, it would have been desirable to assess levels of TIMPs. TIMPs are the natural regulators of MMPs, an effect carried out through an equimolecular binding and inactivation. Both chondrocytes and synovial fibroblasts can produce TIMPs. In this regard, the balance between TIMPs and MMPs is decisive for the outcome (Martel-Pelletier et al., 1994). Functional studies, such as substrate-based zymography, are usually performed to

Fig. 3. Expression of MMPs in rheumatoid synovial fibroblasts exposed to fibrin

Graphs on the left show changes in baseline level expression of the mentioned genes (median SEM), in Ct values using 18s as house keeping gene (the lower Ct values, the higher gene levels are). On the right, representative immunoblots from different experiments are shown. Detection of MMP-1 was done in cell lysates and MMP-3 was

regards the impact of these finding in the erosive tendency.

demonstrate active proteolysis.

Fig. 2. Immune-staining of fibrin and MMPs in the rheumatoid synovial tissues

Left column shows a low-magnification view of a fibrin-rich area of the synovial tissue, with the different staining techniques. Middle column shows the intimal layer of a fibrin-rich area in detail. Right column depicts the staining of the molecules at areas of invasion into bone and cartilage. Fibrin is shown in green (with red counterstaining), MMP-3 is shown in brown (and counterstained in blue), and MMP-1 is shown in blue (with red counterstaining).

#### **2.2.3 Fibrin increased the production of MMP-1 by rheumatoid synovial fibroblasts**

Six different cell cultures were employed in immunoblot experiments. In all cases, treatment with fibrin increased MMP-1 production. In our conditions, both the proenzime and the active form of MMP-1 increased between 18 h and 24 h in rheumatoid synovial fibroblasts treated with fibrin (Figure 3). At 24 h, the active enzyme increased to 4-fold as compared to untreated cells (range: 1,5 to 6; p < 0.03). On the other hand, we could not detect MMP-3 in cell lysates, but it was abundantly found in supernatants from cell cultures. Upon stimulation with fibrin, baseline levels of MMP-3 in supernatants were found increased only in half of the cell lines tested (ns) (Figure 3).

#### **2.3 Discussion and future research**

Our studies illustrate how deposition of fibrin can contribute to the invasive process in rheumatoid arthritis. Previous works had described the distribution of MMP-1 and MMP-3 in the rheumatoid synovial tissue. In the same way, fibrin distribution in rheumatoid joints

Fig. 2. Immune-staining of fibrin and MMPs in the rheumatoid synovial tissues

counterstained in blue), and MMP-1 is shown in blue (with red counterstaining).

in half of the cell lines tested (ns) (Figure 3).

**2.3 Discussion and future research** 

Left column shows a low-magnification view of a fibrin-rich area of the synovial tissue, with the different staining techniques. Middle column shows the intimal layer of a fibrin-rich area in detail. Right column depicts the staining of the molecules at areas of invasion into bone and cartilage. Fibrin is shown in green (with red counterstaining), MMP-3 is shown in brown (and

**2.2.3 Fibrin increased the production of MMP-1 by rheumatoid synovial fibroblasts**  Six different cell cultures were employed in immunoblot experiments. In all cases, treatment with fibrin increased MMP-1 production. In our conditions, both the proenzime and the active form of MMP-1 increased between 18 h and 24 h in rheumatoid synovial fibroblasts treated with fibrin (Figure 3). At 24 h, the active enzyme increased to 4-fold as compared to untreated cells (range: 1,5 to 6; p < 0.03). On the other hand, we could not detect MMP-3 in cell lysates, but it was abundantly found in supernatants from cell cultures. Upon stimulation with fibrin, baseline levels of MMP-3 in supernatants were found increased only

Our studies illustrate how deposition of fibrin can contribute to the invasive process in rheumatoid arthritis. Previous works had described the distribution of MMP-1 and MMP-3 in the rheumatoid synovial tissue. In the same way, fibrin distribution in rheumatoid joints was well known. A novel finding is that all three followed a similar pattern of distribution. We could also confirm their abundance at CPJs, although we cannot draw conclusions as regards the impact of these finding in the erosive tendency.

Our studies suggest that fibrin triggers the transcription of several MMPs and the production of MMP-1. However, the study of MMPs is complex. These molecules are secreted in the form of zymogens, and need in situ activation through the cleavage of an Nterminus peptide. This post-translational modification probably constitutes a major checkpoint for the regulation of MMPs. Additionally, to find out what the global matrix turn-over could be, it would have been desirable to assess levels of TIMPs. TIMPs are the natural regulators of MMPs, an effect carried out through an equimolecular binding and inactivation. Both chondrocytes and synovial fibroblasts can produce TIMPs. In this regard, the balance between TIMPs and MMPs is decisive for the outcome (Martel-Pelletier et al., 1994). Functional studies, such as substrate-based zymography, are usually performed to demonstrate active proteolysis.

Fig. 3. Expression of MMPs in rheumatoid synovial fibroblasts exposed to fibrin

Graphs on the left show changes in baseline level expression of the mentioned genes (median SEM), in Ct values using 18s as house keeping gene (the lower Ct values, the higher gene levels are). On the right, representative immunoblots from different experiments are shown. Detection of MMP-1 was done in cell lysates and MMP-3 was

Innate Mechanisms of Synovitis – Fibrin Deposits Contribute to Invasion 125

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studied in the extracellular fraction. Both antibodies detected both the zymogen (upper band) and the active protease (lower band).

Nevetheless, it was interesting to find that three MMPs previously correlated to the invasive potential of rheumatoid synovial fibroblasts, were up-regulated by fibrin at the mRNA level (Tolboom et al., 2002). Due to its insolubility, fibrin might be degraded in a non-constitutive way by MMP-3 secreted by surrounding cells (Bini et al., 1999). On the balance of the evidence, we believe that fibrin-rich regions should not be considered a result of longstanding inflammation, but a site for active destruction.

In agreement with previous studies, most of the synovial fibroblast cultures that we employed in our studies did not constitutively express MMP-13 as assessed with qPCR techniques, and only in some was it induced after exposure to fibrin (Moore et al., 2000). In the production of this, as well as the other proteases tested, there seemed to be a high variability between patients. In fact, our studies suggested the existence of two subsets according to their response to fibrin. Approximately half of the cultures strongly reacted with the up-regulation of MMPs, while the other half showed mild or absent response. We believe that this is another example of the heterogeneous character of rheumatoid arthritis.

Although there was no regulation of MT1-MMP at the mRNA level by fibrin, an interesting finding drawn by our experiments was the high expression of MT1-MMP by unstimulated rheumatoid synovial cells, pointing to the prominent role of the protease in the activity of synovial fibroblasts as already suggested (Miller et al., 2009).

In summary, using a novel culture system for the study of fibrin interaction with synovial cells, we could show induction of proteases putatively associated to invasiveness, that were further localized at fibrin-rich areas in the synovial tissues.
