Cell-culture media (RPMI 1640 and M199), FCS, penicillin, streptomycin, amphotericin B, Taq DNA polymerase, dNTPs, and primers were from Invitrogen (Cergy-Pontoise, France). Cell-culture media never had an endotoxin level above 0.04 ng/mL, as tested by the Limulus chromogenic assay. LPS from Escherichia coli O55:B5, polymyxin B, and type XI collagenase were obtained from Sigma (Saint Quentin Fallavier, France). The First Strand cDNA synthesis kits and ³²P dATP were from Amersham Pharmacia Biotech (Saclay, France). A Nucleospin RNA II extraction kit was from Macherey-Nagel (Souffelweyersheim, France). Atlas human cancer cDNA expression arrays were from Clontech (Ozyme, Saint Quentin Yvelines, France). BINDAZYME™ ProMMP-3 enzyme immunoassay kit was from The Binding Site (Saint Egreve, France). The SYBR^®Green PCR master mix was from Applied Biosystems (Courtaboeuf, France). Buffers were prepared with apyrogenic water obtained from Braun Medical (Boulogne, France).

Human FLSs were isolated from RA synovial tissues from three patients at the time of knee joint arthroscopic synovectomy, as described previously 22. The diagnoses conformed to the revised criteria of the American College of Rheumatology 23. Human FLSs were also isolated from osteoarthritis (OA) synovial tissues from three patients who were having joint replacements. FLS cultures were performed as previously described 20. Briefly, tissues were minced, digested with 1 mg/mL collagenase in serum-free RPMI 1640 for 3 hours at 37°C, centrifuged (130 g for 10 minutes at 4°C) and resuspended in M199-RPMI 1640 (1:1) containing 2 mm l-glutamine, penicillin (100 IU/mL), streptomycin (100 μg/mL), amphotericin B (0.25 μg/mL), and 20% heat-inactivated FCS (complete medium). After overnight culture, nonadherent cells were removed and adherent cells were cultured in complete medium. At confluence, cells were trypsinized and passaged in 75-cm² culture flasks in complete medium containing 10% heat-inactivated FCS. Experiments were performed between the third and the ninth passages, during which time cultures were a homogeneous population of fibroblastic cells, negative for CD16 as determined by FACS analysis. Before activation experiments, cells were deprived of serum for 24 hours, and then the appropriate stimuli diluted in serum-free RPMI 1640 with antibiotics were added. To eliminate the possibility that the observed effects were due to LPS contamination, all the experiments were performed in the presence of polymyxin B (2 μg/ml). Cell numbers and cell viability were assessed using the MTT test (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test) as described elsewhere 24.

Recombinant protein I/II of Streptococcus mutans OMZ 175 was purified from pHBsr-1 transformed E. coli cell extract by gel filtration and immunoaffinity chromatography as described elsewhere 25. The purity of the protein was checked by SDS–PAGE after staining with Coomassie blue. Protein I/II migrated as a single band having an apparent molecular weight of 195 kDa.

FLSs (3 ×10⁶ cells) were stimulated with 600 μL of serum-free RPMI 1640 containing protein I/II (125 pm final concentration). After a 4-hour incubation period, cells were centrifuged (130 g for 10 min at 4°C), and total RNA was extracted from cell pellets using the Nucleospin RNA II extraction kit in accordance with the manufacturer's instructions.

FLSs (5 ×10³ cells) were grown to confluence in 96-well plates (7–10 days) and then stimulated with 200 μL of serum-free RPMI 1640 containing protein I/II (125 pm final concentration). After an 18-hour incubation period, a heterologous two-site sandwich ELISA was used to estimate pro-MMP-3 release in the culture supernatants.

The gene expression profile was examined using Atlas human cancer cDNA expression arrays, consisting of nylon membranes spotted in duplicate with cDNA fragments of 588 known genes. These genes are classified into several functional groups, such as oncogenes and tumor suppressors, growth factors and receptors, and regulators of cell adhesion, angiogenesis, and cell cycle (listed at http://www.bdbiosciences.com/clontech/atlas/genelists/index.shtml). Total RNA (2 μg) was converted into ³²P-labeled first-strand cDNA following the protocol provided by the manufacturer. Briefly, total RNA was reverse-transcribed using MMLV (Moloney murine leukemia virus) reverse transcriptase and ³²P-labeled dATP in a PCR thermal cycler set at 50°C for 25 min. Labeled cDNA probes were then purified from unincorporated ³²P-labeled nucleotides and small (<0.1 kb) cDNA fragments using column chromatography. Probes (2–10 × 10⁶ cpm) were freshly applied to cDNA array membranes to hybridize overnight at 68°C in continuously agitated roller bottles. After hybridization, membranes were washed four times, for 30 min each, at 68°C with 2 × SSC (standard saline citrate), 1% SDS, and once with 0.1 × SSC, 0.5% SDS, followed by one wash with 2 × SSC at room temperature. Array membranes were wrapped in plastic and exposed to a phosphor screen for 1–5 days, depending on the radiation intensity of the bound fragments. After image acquisition on a Storm phosphor imager (ImageQuant, Molecular Dynamics, Sunnyvale, CA, USA), spots were quantified using the AtlasImage 2.0 Software (Clontech), developed specifically for analysis of the Atlas cDNA expression arrays. All spots were individually checked by hand to ensure accuracy of the detection method. A dot was considered to be positive if it was well located and at least three times the local background level. Spots of poor quality were not included. For each patient, the relative expression level of each gene between protein-I/II-stimulated and control FLSs was evaluated and standardized based on expression levels of housekeeping genes included on each array.

Total RNA (2 μg) isolated from FLSs was reverse-transcribed using the First Strand cDNA Synthesis Kit in accordance with the manufacturer's instructions. Total RNA (8 μL) was mixed with 5 μl of the bulk reaction mix, 1 μl of DTT (200 mm), and 1 μl of the Not I-d(T)₁₈ bifunctional primer (0.2 μg/ml). The reaction was carried out for 1 hour at 37°C. Real-time PCR was performed in 96-well plates in a total volume of 25 μl using the SYBR^®Green PCR master mix (containing SYBRGreen dye, AmpliTAq Gold^® DNA Polymerase, dNTPs with dUTP, passive reference and optimized buffer components) and gene-specific primers (250 nm): MMP-3 1) 5' GCA GTT TGC TCA GCC TAT CC 3' and 2) 5' GAG TGT CGG AGT CCA GCT TC 3' 26; GAPDH (glyceraldehyde-3-phosphate dehydrogenase), 1) 5' AGC AAT GCC TCC TGC ACC ACC AAC 3' and 2) 5' CCG GAG GGG CCA TCC ACA GTC T 3' 27. After incubation at 50°C for 10 min and at 95°C for 10 min, samples were subjected to 40 rounds of amplification for 15s at 95°C, 15s at 56°C, and 40s at 72°C using the AbiPrism 7700 Sequence Detection System (Applied Biosystems). Amplification products were detected as an increased fluorescent signal of SYBR^®Green during the amplification cycles. Results were obtained using SDS Software (Applied Biosystems, Foster City, CA, USA) and evaluated using Excel (Microsoft). Primer efficiency was calculated for MMP-3 and GAPDH using the standard curve method in accordance with the supplier's recommendations. As the MMP-3 and GAPDH amplifications were about equally efficient, the relative expression levels of the MMP-3 gene were evaluated using the 2^-ΔΔCT method as described by Applied Biosystems. Additionally, to confirm the amplification specificity of each gene product, the PCR products were subjected to a melting-curve analysis.

Pro-MMP-3 levels in cell-culture supernatants were determined using the BINDAZYME™ pro-MMP-3 Enzyme Immunoassay Kit in accordance with the manufacturer's instructions. Briefly, duplicate samples were added to wells coated with anti-pro-MMP-3 and were then incubated with anti-pro-MMP-3 peroxidase conjugate; the peroxidase substrate (TMB) was then added. After the reaction had been stopped by the addition of phosphoric acid (3 m), the optical density of each well was read at 450 nm. Pro-MMP-3 levels were calculated using a standard curve.

Values are represented as means ± standard error of the mean. The significance of the results was analyzed using Student's two-tailed t-test. Values of P < 0.05 were considered significant.

In this study we used a gene array technique to further investigate the cellular response of FLSs to protein I/II. As we were particularly interested in genes that could contribute to the aggressive behavior of RA FLSs, we chose Atlas human cancer cDNA expression arrays containing genes involved in cell-cycle regulation, cellular adhesion, and inflammation. In parallel to RA FLSs, we used OA FLSs in order to study the protein-I/II-induced response of FLSs isolated from a noninflammatory arthropathy. After a 4-hour incubation in the presence or absence of 125 pm protein I/II, the total RNA from FLSs of three RA and three OA patients was extracted, and radiolabeled cDNA probes were subsequently hybridized to the cDNA arrays. Figure 1 shows the expression patterns of FLSs isolated from one RA and one OA patient in nonstimulated controls after 4 hours (Fig. 1a,1c) and after stimulation with protein I/II for 4 hours (Fig. 1b,1d). The products of some differentially expressed genes (e.g. the gene for IL-6) are easily observed. The relative expression level of each gene in protein-I/II-stimulated FLSs versus control FLSs was evaluated with the AtlasImage 2.0 Software.

When a cut-off of a threefold change in mRNA expression was used, 28 genes were up-regulated and 5 genes were down-regulated in a least one of the three RA FLSs tested (Tables 1 and 2). These modulated genes account for about 6% of all profiled genes. Among the genes up-regulated by protein I/II in all three RA FLSs, two genes encoding proinflammatory cytokines, IL-6 and leukemia inhibitory factor, showed a strong expression level. The expression of other cytokine genes included on the array, such as TNF-α or IL-1, was not affected by protein I/II stimulation (IL-8 was not represented on the array). Protein I/II also strongly up-regulated the expression of MMP-3 (stromelysin 1), whereas the expression of genes encoding other members of the MMP family was not modified. Furthermore, a transcription factor, interferon regulatory factor 1 (IRF-1), as well as a growth factor, fibroblast growth factor-5 (FGF-5), were up-regulated by protein I/II in the three RA FLSs, although the expression levels were lower. Besides this group of genes which were strongly up-regulated in all RA FLSs, the expression of other genes was more heterogeneous and varied between the different RA FLS cultures. Among these genes, mainly involved in protein turnover and in cell signaling and communication, two genes, encoding CDC27Hs protein and interferon γ antagonist, were strongly up-regulated and the expression of three more genes (those encoding integrin αE, vimentin, and transmembrane protein SEX) was less intensely up-regulated in two of the three RA FLSs.

Interestingly, the expression of a few genes was down-regulated by protein I/II and only one gene was down-regulated in all three RA FLSs tested, namely, that for insulin-like growth factor binding protein-4. This member of the family of insulin-like growth factor binding proteins, which act as carriers and regulators of insulin-like growth factor, is believed to play an important role in maintaining the equilibrium between synthesis and degradation of tissue matrix molecules.

In OA FLSs, the number of genes modulated within 4 hours by protein I/II was much lower than in RA FLSs: protein I/II up-regulated the expression of only one gene, namely IL-6, in all OA FLSs tested. Other genes were up-regulated but with a high variability among FLSs cultures (Table 3). A very interesting finding was that one of the three OA FLS cultures (from patient 3) showed a gene expression pattern that was very close to the patterns obtained with protein-I/II-stimulated RA FLSs. Clinical investigations revealed that this patient had a secondary arthropathy of the knee occurring during a long remission phase of RA. This patient was therefore excluded from further experiments.

The up-regulation of IL-6 gene expression found in both RA and OA FLSs in response to protein I/II has been previously shown to be followed by an increase of IL-6 release from these cells (MB Zeisel and colleagues, unpublished data) 20. Since we were primarily interested in genes that could contribute to the aggressive, invasive behavior of RA FLSs, the marked up-regulation of the MMP-3 gene, which plays a major role in cartilage degradation, prompted us to further study its expression in FLSs stimulated with protein I/II.

In order to confirm the up-regulation of the expression of MMP-3 gene in RA FLSs by protein I/II, we performed real-time PCR analysis for MMP-3. Total RNA was extracted from FLSs isolated from three RA FLSs stimulated with protein I/II or left untreated, reverse-transcribed, and then amplified by real-time PCR. MMP-3 mRNA showed a mean 36-fold up-regulation after protein I/II stimulation of RA FLSs in comparison with control cells, confirming the results obtained with the cDNA array experiments.

To determine whether the up-regulation of the expression of MMP-3 gene by protein I/II stimulation in RA FLSs resulted in changes in gene translation and thereby contributed to an increase in the release of pro-MMP-3, we next measured the level of pro-MMP-3 in culture supernatants from control and RA FLSs that had been stimulated with protein I/II for 18 hours. As MMP-3 is first synthesized as a proenzyme (pro-MMP-3), which is subsequently cleaved to generate active MMP-3, we chose to assay pro-MMP-3 levels, as the activation process might not occur in our in vitro cultures. Figure 2 shows that control RA FLSs basally released pro-MMP-3 (1.4 ± 0.4 ng/mL) and that stimulation with protein I/II induced an increase in pro-MMP-3 secretion (3.1 ± 0.4 ng/mL, P < 0.01). Control OA FLSs basally released pro-MMP-3, but protein I/II stimulation did not significantly increase pro-MMP-3 secretion from these cells (Fig. 2). To confirm the activation of FLSs, we assayed IL-6 and IL-8 levels in the same supernatants used for pro-MMP-3 assay, and found that protein I/II induced a significant increase in the release of these cytokines from both RA and OA FLSs (data not shown).