The Immunosuppressant FTY720 (Fingolimod) enhances Glycosaminoglycan depletion in articular cartilage

FTY720-P was kindly donated by Novartis Pharma AG (Basel, Switzerland) and dissolved in DMSO-HCL. Bovine IL-1β and TNF-α were purchased from AbD Serotec(Oxford, UK). XG076 was purchased at Calbiochem (Darmstadt, Germany). DMEM High Glucose with L-Glutamine, DMEM/Ham's F-12 with L-Glutamine 1:1, FCS and Penicillin/Streptomycin solution was purchased at PAA Laboratories (Pasching, Austria). We acquired iNOS antibodies (Upstate, Lake Placid, NY) and antibodies for actin (Sigma-Aldrich, St. Louis, MO). Secondary antibody was purchased from Cell Signaling (Danvers, MA).

Cartilage was harvested from bovine metacarpophalangeal joints of adult cows (20 - 24 months, n = 21) under aseptic conditions. Cartilage tissue was minced and digested in 0.2% collagenase B (F. Hoffman La Roche Ltd., Basel, Switzerland) for 16 hours. The resulting cell suspension was filtered through a nylon mesh with pores of 70 μm (BD Pharma, Le Pont-De-Claix, France). Cells were counted and viability tested using trypan blue dye (Sigma-Aldrich). Bovine chondrocytes were then cultured in monolayer at 37°C, 5% O2 and 5% CO2, in DMEM/F-12 1:1 supplemented with 10% FCS and 1% Penicillin/Streptomycin solution over 1 passage. Upon 80%-90% confluence cultures were incubated with serum free medium 24 h prior to experiments. Chondrocyte viability in the presence of 0.01 μM to 10 μM FTY720-P, the corresponding amount of vehicle (DMSO-HCL) and 100 μM XG076 was assessed for 72 h using a MTS ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay (Promega, Madison, WI) according to the manufacturers protocol. MTS assay was carried out in hexaplicates and repeated three times using chondrocytes from 3 different animals

Three independent experiments using chondrocytes from 3 different animals were performed in duplicates for each treatment group. Chondrocytes were allowed to grow over one passage and upon 80%-90% confluence cultures were serum-depleted 24 h prior to experiments. Chondrocytes were then treated with 10 ng/ml IL-1β or 100 ng/ml TNF-α in combination with 0.1 to 3 μM FTY720-P or vehicle solution for 3 h. RNA was isolated using Tri-reagent (Sigma-Aldrich, St. Louis, MO) according to the manufacturers recommended protocol. To avoid potential DNA contamination, DNA was digested with 1 U DNase (Fermentas, Burlington, ON) per μg RNA for 30 minutes at 37°C. A total of 1 μg of RNA was then reverse transcribed using RevertAid cDNA Synthesis Kit (Fermentas) and random hexamer primers according to the provided manual. For amplification all samples were run in triplicates using a master mix containing SYBR green (2× SYBR Green PCR Master Mix, Invitrogen Corp., Carlsbad, CA). Primers (MWG, Ebersberg, Germany) were designed with Primer3 software [14]. Sequences of primers are listed in additional file 1, Table S1. For normalization the housekeeping genes Hydroxymethyl-bilane synthase, Beta-2-microglobulin, Beta actin, Hypoxanthine phosphoribosyltransferase 1, Ribosomal protein S18 and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were evaluated in our experimental setting. GAPDH showed the least variation over all treatments and was therefore chosen as housekeeping gene for the following analyses. The relative amount of cDNA was calculated using ABI prism sequence detecting software (Applied Biosystems) based on a standard curve, according to the manufacturers' manual. Minimum information for publication of quantitative real-time PCR experiments is supplied in additional files 1 and 2[15].

Three independent experiments using chondrocytes from 3 different animals were performed. Chondrocytes were allowed to grow over one passage and upon 80%-90% confluence cultures were serum-depleted 24 h prior to experiments. Total protein was extracted from cultured chondrocytes after 24 h of treatment with10 ng/ml IL-1β in combination with 3 μM FTY720-P or vehicle solution, using lysis buffer (50 mM Tris-HCL, 4.1 M NaCl, 0.1% Triton X-100, 5 mM EDTA, 1% protease inhibitor cocktail; all Sigma Aldrich). After brief sonication protein concentration was measured with DC Protein assay (BioRad). 7 μg of protein were separated by SDS-PAGE using a 10% polyacrylamide gel and were transferred to a nitrocellulose membrane (BioRad, Hercules, CA). After overnight blocking in 5% skim milk in Tris-buffered saline the membranes were incubated with primary antibodies against iNOS (1:2000) and actin (1:5000) for 2 h. Thereafter membranes were rinsed in blocking solution and incubated for 1 h with secondary antibody conjugated to horseradish peroxidase (1:3000). Bands were visualized using ECL plus detection system (Amersham, Arlington Heights, IL).

Three independent experiments were performed using cartilage from 3 different animals. Cartilage explants of 4 × 4 × 2 mm (wet weight 13,3 mg ± 0.9 mg) were harvested from bovine metacarpophalangeal joints under aseptic conditions and cultured at 37°C, 5% O2 and 5% CO2, in DMEM/F-12 1:1 supplemented with 10% FCS and 1% Penicillin/Streptomycin solution for 24 h. After another 24 h in the absence of FCS cartilage explants were treated with 0.5-3 μM FTY720-P or vehicle control and 10 ng/ml IL-1β or 100 ng/ml TNF-α over 5 days. Medium was changed and treatment renewed on day 3. Treatment was carried out in triplicates for each treatment group. Thereafter explants were weighted and digested in phosphate buffer (2.76 g/l NaH2PO4, 0.38 g/l EDTA, 0.25 g/l DTT all Sigma-Aldrich, pH 6.8) containing 0.6 g/l papain (Sigma-Aldrich). GAG concentration in culture supernatants of day 3, day 5 and the digested explants was determined by 1,9-dimethylene blue (DMB; Sigma-Aldrich) reaction [11, 16]. In brief 20 μl of the samples and chondroitin sulphate (Sigma-Aldrich) standard dilution were incubated with 180 μl DMB solution (38.5 μM DMB, 40 mM NaCl, 40 mM glycin, 0.5% Ethanol, pH 3) for 5 minutes. Absorbance at 530 nm was measured with Spectra max 384-plate reader. Results were normalized to wet weight of the explants.

Three independent experiments using cartilage from 3 different animals were performed. Bovine cartilage e xplants were harvested as described above and were cultured at 37°C, 5% O2 and 5% CO2, in DMEM/F-12 1:1 supplemented with 1%Penicilin/Streptomycin solution. Media containing either 10 ng/ml IL-1β or 100 ng/ml TNF-α in combination with 3 μM FTY720-P or vehicle solution was changed on the 3^rd and 6^th day. Upon 8 days of treatment, cartilage explants were fixed in 4% formalin solution for 24 hours and then embedded in paraffin. Sections of 3 μm were stained with 0.1% safranin O solution, 0.001% fast green solution and Weigert's iron hematoxylin solution [17].

Results are presented as mean ± standard error of the mean (SEM). Real-time RT-PCR results are expressed as mean ± SEM percentage over control. Normal distribution of the data was assessed using Kolmogorov-Smirnov test. Data in all treatment groups were normally distributed. Data were analyzed by one-way ANOVA followed by post-hoc analysis using Bonferroni corrected t-test. SPSS 13.0 (IBM, Chicago, IL) was used for statistical analysis. Differences between two groups were considered significant at the P < 0.05 level.

Chondrocyte viability was unchanged in the presence of up to 10 μM FTY720-P or 100 μM of the MMP-3 inhibitor XG076 (P > 0.05, data not shown). Treatment of chondrocytes with 10 ng/ml interleukin-1 (IL-1)β resulted in 18-fold up-regulation of iNOS mRNA expression (P < 0.05). Co-treatment with up to 3 μM FTY720-P reduced iNOS mRNA expression by 40 ± 8% (mean ± SEM, P < 0.05) compared to vehicle solution. (Figure 1A)

Treatment with 100 ng/ml tumor necrosis factor (TNF)-α led to a 18-fold (P < 0.05) up-regulation of iNOS mRNA. Co-treatment with 3 μM of FTY720-P reduced TNF-α induced iNOS expression by 47 ± 16% (P < 0.05). A similar trend was observed when IL-1β stimulated chondrocytes were co-treated with unphosphorylated FTY720 (data not shown). The maximal effect was observed later (after 6 h) and was less pronounced compared to treatment with FTY720-P.

iNOS protein expression was significantly enhanced after 24 h of treatment with IL-1β (24-fold, P < 0.01) or TNF-α (11-fold, P < 0.01). As expected from RT-PCR results, co-treatment with 3 μM of FTY720-P reduced IL-1β and TNF-α induced iNOS protein expression by 39%, (P < 0.05) and 69.4% (P < 0.05) respectively (Figure 1B and 1C).

Culturing cartilage explants for 8 days with 0.1-3 μM of FTY720-P did not result in altered depletion of GAG compared to vehicle treated cartilage (Figure 2G). IL-1β induced a significant increase in GAG depletion (Figure 2H). Untreated cartilage explants lost 5.9 ± 1.3 μg GAG/mg wet weight over 5 days compared to 23.7 ± 3.9 μg/mg lost by IL-1β treated explants (P < 0.05). Furthermore co-treatment with FTY720-P resulted in significantly higher amounts of GAG loss. Co-treatment of IL-1β with 3 μM of FTY720 increased GAG depletion to 38.4 ± 4.3 μg/mg (P < 0.05 compared to IL-1 treatment). Treating cartilage explants with TNF-α led to a GAG depletion of 12.3 ± 1.0 μg/mg, which was not significantly different from untreated explants (7.5 ± 2.1 μg/mg, P = 0.18). Upon incubation with FTY720-P a significant increase in GAG depletion was observed. 3 μM of FTY720-P led to 31.2 ± 6.2 μg/mg (P < 0.01 vs. TNF-α) of GAG depleted from cartilage explants (Figure 2H).

These results were confirmed by histology in an independent series of experiments. GAG content as determined by safraninO staining in FTY720-P treated cartilage was not different from untreated cartilage (Figure 2A and 2B). IL-1β treatment resulted in considerable discoloration of cartilage. Co-treatment with 3 μM of FTY720-P further enhanced the effect (Figure 2C and 2D). Again TNF-α treatment showed a mild negative effect on cartilage staining. Combination with FTY720-P led to discoloration of the upper half of the cartilage explants. This effect was independent of whether cartilage explants were placed in the culture wells correctly or up-side down (Figure 2E and 2F).

As MMP-3 is crucial for activation of pro-MMP's, we used XG076, an inhibitor of pro-MMP-3 activation. Upon inhibition of pro-MMP-3 with XG076 the effect of IL-1β and TNF-α on GAG depletion was rendered to levels of the untreated controls. Furthermore co-treatment with 3 μM FTY720-P did not result in increased GAG depletion (Figure 3A).

To asses which protease was responsible for the enhanced GAG depletion by FTY720-P we examined gene expression of MMP-1,-3.-13, ADAMTS-4,-5 and COX-2. Treatment of chondrocytes with either IL-1β or TNF-α led to a 5.5-fold and 8.4-fold up-regulation of MMP-13 mRNA, respectively (both with P < 0.01). Co-treatment with FTY720-P reduced MMP-13 mRNA expression by 52 ± 8% and 74 ± 9% (both P < 0.05, Figure 3B)

In contrast to the effect of FTY720-P on MMP-13, MMP-3 and ADAMTS-5 expression induced by IL-1β was further enhanced by FTY720-P, however not reaching statistical significance (P = 0.063 and 0.054 respectively; Figure 3C and 3D). Expression of COX-2, ADAMTS-4 and MMP-1 was enhanced by TNF-α and IL-1β treatment. No effect was observed upon co-treatment with FTY720-P (data not shown).