Key regulatory molecules of cartilage destruction in rheumatoid arthritis: an in vitrostudy

The local ethical committee of the Charité Berlin approved this study.

For chondrocyte isolation, human articular chondrocytes from six normal donors post mortem without obvious joint defects and macroscopic signs of osteoarthritis were isolated from the medial and lateral condyle of femur bones obtained from the Institute of Pathology at the Charité University Hospital Berlin. The average patient age was 60 years, ranging from 39 to 74 years. Chondrocytes were harvested as described previously [25] and expanded in monolayer culture with RPMI 1640 medium (Biochrom, Berlin, Germany) supplemented with 10% human serum, 100 ng/ml amphotericin B (Biochrom), 100 U/ml penicillin and 100 μg/ml streptomycin (Biochrom). Throughout the experiment, the same pool of human serum (n = 5 donors) was used. Medium was changed every 2 to 3 days. Reaching subconfluence, chondrocytes were detached with 0.05% trypsin and 0.02% EDTA (Biochrom) and cryopreserved. After cryopreservation, human chondrocytes were expanded in a monolayer and, after reaching subconfluence again, the cells were trypsinized and subsequently immobilized in alginate beads.

Human SV40 T-antigen immortalized SF were derived from a patient with RA (HSE cell line; RASF) and from a normal donor (K4IM cell line; NDSF), respectively. Synovial pannus tissue from a patient with RA was obtained by surgical synovectomy of the knee joint from a patient diagnosed according to the American College of Rheumatology revised criteria as having active RA [26]. Normal donor synovial tissue was obtained during meniscectomy from a 41-year old male suffering from a meniscus lesion [22]. After isolation of the human synovial fibroblasts, the cells were transfected with SV40 TAg expression vector, yielding immortalized synovial fibroblast cell lines [22, 26]. Immortalized synovial fibroblasts derived from the patient with RA represent RASF, and immortalized synovial fibroblasts derived from the normal donor patient represent NDSF. SF were expanded in a monolayer with RPMI 1640 medium supplemented with 10% human serum, 100 U/ml penicillin and 100 μg/ml streptomycin. Medium was changed every 2 to 3 days.

Alginate (Sigma, Taufkirchen, Germany) solution was prepared in 150 mM NaCl and 30 mM HEPES at 3% (w/v) and sterilized by autoclaving. Equal volumes of alginate solution and human articular chondrocyte suspension were combined to yield suspensions with final cell densities of 2 × 10⁷ cells/ml in 1.5% (w/v) alginate. Spherical beads were created by dispensing droplets of alginate cell suspension from the tip of an 18-gauge needle into a bath of 120 mM CaCl₂, 10 mM HEPES, 0.01% Tween 80 and 150 mM NaCl followed by gelation for 20 minutes. Beads were cultured in batches in six-well plates for 2 weeks in RPMI 1640 medium supplemented with 10% human serum, 100 ng/ml amphotericin B, 100 U/ml penicillin, 100 μg/ml streptomycin and 170 μM l-ascorbic acid 2-phosphate (Sigma).

Medium of NDSF and RASF at 80% confluence was conditioned for 48 hours, and supernatants were adjusted to the same ratio of volume/cell number and stored at -20°C. After 2 weeks of three-dimensional chondrocyte cultivation in alginate beads, medium of cartilage-like beads was replaced by collected supernatants of NDSF (NDSFsn) or RASF (RASFsn). Interactive cultivation was performed for 48 hours (Figure 1). To determine baseline gene expression, a control group of alginate-embedded chondrocytes was treated with cultivation medium for 48 hours.

Isolation of total RNA was performed for the six different stimulated donor chondrocytes separately. Afterwards, equal amounts of total RNA from three stimulated donor chondrocytes (1.5 μg from each donor) were pooled, yielding two different experimental groups of NDSFsn-stimulated and RASFsn-stimulated chondrocytes and of unstimulated chondrocytes. From each experimental group, 2.5 μg of combined total RNA was used for microarray applications and 2 μg was used for real-time RT-PCR. Gene expression profiling from pooled RNA samples derived from individual donors with a reasonable replication of pooled arrays has recently been determined to be statistically valid, efficient and cost-effective [27, 28].

Microarray analyses of RASFsn-stimulated and NDSFsn-stimulated chondrocytes and unstimulated chondrocytes were performed for two experimental groups (n = 2). The Human Genome U133A GeneChip (Affymetrix, High Wycombe, UK) that determines the expression level of 18,400 transcripts and variants representing about 14,500 human genes was used for gene expression analysis. Microarray preparation was performed in accordance with the manufacturer's protocol. In brief, equal quantities of high-quality total RNA from experimental groups (2.5 μg of each) were reverse transcribed to single-stranded cDNA. After a second-strand cDNA synthesis, biotin-labeled antisense cRNA was generated by in vitro transcription. Next, 15 μg of each generated cRNA preparation was fragmented and hybridized to the oligonucleotide microarray. Washing, staining and scanning were performed automatically with the Affymetrix GeneChip System. Raw expression data were analyzed using (1) GeneChip Operating Software (GCOS) version 1.2 (Affymetrix) in accordance with the manufacturer's recommendations and (2) Robust Multi-array Analysis version 0.4α7 (RMA) [29]. Differentially expressed genes reproducibly showed a fold change of ≤-2 (decrease) or a fold change of ≥2 (increase) as determined by GCOS and RMA data processing. The filtered gene list was functionally annotated with the use of reports from the literature. Hierarchical cluster analysis with signal intensity of differentially expressed genes and the Pearson correlation distance were performed with Genesis 1.7.2 software [30]. Microarray data have been deposited in NCBIs Gene Expression Omnibus (GEO) and are accessible through GEO series accession number GSE10024.

Equal quantities of high-quality total RNA from both experimental groups (2 μg of each) of both NDSFsn-stimulated and RASFsn-stimulated chondrocytes were reverse transcribed with iScript cDNA synthesis kit (Bio-Rad, Munich, Germany) in accordance with the manufacturer's instructions. TaqMan real-time RT-PCR was performed in triplicates in 96-well optical plates on an ABI Prism 7700 Sequence Detection system (Applied Biosystems, Darmstadt, Germany) with Gene Expression Assays for TaqMan probes and primer sets, which were pre-designed and pre-optimized by Applied Biosystems. Quantitative gene expression was analyzed for chemokine (C-X-C motif) receptor 4 (CXCR4, assay ID Hs00607978_s1), thioredoxin interacting protein (TXNIP, Hs00197750_m1), chondroitin sulfate proteoglycan 2 (CSPG2, Hs00171642_m1), IFN-α inducible protein-6–16 (IFI-6–16, Hs00242571_m1), cyclooxygenase-2 (COX-2, Hs00153133_m1), cartilage oligomeric matrix protein (COMP, Hs00164359_m1), steroid sulfatase (STS, Hs00165853_m1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Hs99999905_m1). The expression levels of selected differentially expressed genes were normalized to endogenous glyceraldehyde-3-phosphate dehydrogenase expression level and calculated with the 2^-ΔΔCtformula (ABI Prism 777 Sequence Detection System User Bulletin no. 2). For statistical analysis, Students' ttest was applied.

The human protein membrane array (RayBiotech, Norcross, GA, USA) simultaneously profiles 30 custom proteins in duplicate. Experiments were performed in accordance with the manufacturer's instructions. In brief, conditioned supernatants of both NDSF and RASF were adjusted with medium to the same ratio of volume/cell number and stored at -20°C. Human cytokine array membranes were incubated for 30 min in 2 ml of blocking buffer and afterwards for 2 hours in 2 ml of sample supernatant at 20°C. After being washed, the membranes were incubated with biotin-conjugated antibodies (1:250 dilution; 1 ml per array membrane) at room temperature for 2 hours and washed again. A solution containing horseradish peroxidase-conjugated streptavidin (1:1,000 dilution; 2 ml) was added and incubation was continued for 2 hours followed by a third washing step. Proteins were detected by enhanced chemiluminescence and the membranes were briefly exposed to X-ray films (Amersham, Munich, Germany) for 30 s, 1 min, 2 min and 4 min. Array images were acquired at a resolution of 300 d.p.i. on a computer photo scanner.

Alginate beads were generated reproducibly with a spherical shape and a diameter of 2.13 ± 0.13 mm (data not shown). Differential expression analysis of chondrocytes stimulated with RASFsn and NDSFsn was used to determine molecular RA-related patterns of chondrocyte gene expression. GCOS and RMA statistical analyses showed 68 reproducibly differentially expressed genes; 44 genes were induced (fold change ≥ 2) and 24 genes were repressed (fold change ≤ -2). The differentially expressed genes were functionally annotated with reports from the literature and were classified into six functional groups (Table 1). Visualization of these differentially expressed genes by hierarchical clustering demonstrated that the expression patterns of the corresponding experimental groups for both RASFsn-stimulated and NDSFsn-stimulated chondrocytes were similar to each other; corresponding groups clustered and showed little degree of variability (Figure 2).

Basically, RASFsn-stimulated chondrocytes showed, in comparison with NDSFsn-stimulated chondrocytes, an altered expression of genes associated with inflammation (NF-κB signaling pathway, cytokines/chemokines and receptors, and immune response) and cartilage destruction (matrix metalloproteinases (MMPs), chondrocyte apoptosis, and suppressed matrix synthesis).

As shown in Table 1, genes related to inflammation were differentially expressed in RASFsn-stimulated chondrocytes: cyclooxygenase-2 (COX-2) and phospholipase A₂ group IIA (PLA2G2A) regulating the synthesis of prostaglandins, adenosine A2A receptor (ADORA2A) as an important immuno-modulator of inflammation, and steroid sulfatase (STS) and hydroxysteroid (11-β) dehydrogenase 1 (HSD11B1), which are involved in the biosynthesis of steroid hormones. Moreover, expression of several genes involved in the NF-κB signaling pathway showed differential expression, including interleukin-1 receptor antagonist (IL1RN), receptor-interacting serine/threonine kinase 2 (RIPK2), toll-like receptor 2 (TLR2), spermine synthase (SMS), thioredoxin interacting protein (TXNIP) and BCL2-related protein A1 (BCL2A1). Apart from NF-κB-associated genes, some cytokines/chemokines and receptors were induced, such as granulocyte colony-stimulating factor 3 (CSF3), IL-23A and hepatocyte growth factor receptor (Met), the chemokines CXCL1–3 (Groα–γ), CXCL8 (IL-8) and CCL20 (MIP-3β), and the chemokine receptor CXCR4.

Additionally, profiling of gene expression in RASFsn-stimulated chondrocytes showed a repression of genes involved in cell proliferation and differentiation, and a distinct induction of numerous genes associated with immune response, including 2',5'-oligoadenylate synthetase 1 (OAS1), 2',5'-oligoadenylate synthetase-related protein p30 (OASL) and IFI-6–16.

Besides inflammation, RASFsn-stimulated chondrocytes showed a distinct expression of genes associated with cartilage destruction, including chondrocyte apoptosis (BCL2A1, RIPK2 and TLR2) and suppressed extracellular matrix (ECM) synthesis; cartilage oligomeric matrix protein (COMP), chondroitin sulfate proteoglycan 2 (CSPG2) and thrombospondin 2 (THBS2) were repressed in RASFsn-stimulated chondrocytes.

Apart from the 68 differentially expressed genes reaching a fold change of ≥2 or ≤-2, the expression of already established marker genes of cartilage destruction that failed to meet the stringent twofold regulation criteria is listed in Table 2. However, these established RA-related genes showed also differential expression of at least 1.5-fold (GCOS data), including genes involved in oxygen damage and IL-1β, IL-6, prostaglandin E synthase (PGES) and genes associated with NF-κB and TNF-α. Moreover, the expression of the matrix-degrading enzymes MMP10 and MMP12 was induced and the expression of testican-1 and genes encoding numerous collagens was repressed.

Thus, genome-wide microarray data displayed differential expression of distinct genes in human chondrocytes that have already been implicated in inflammatory diseases or cartilage destruction. However, several differentially expressed genes have not yet been described as being regulated in chondrocytes during RA-related destruction of cartilage.

The expression profiles of selected genes obtained by microarray analysis were verified by gene expression analysis with real-time RT-PCR. Because numerous RA-relevant genes were differentially expressed in RASFsn-stimulated chondrocytes, representative candidate genes associated with inflammation and cartilage destruction were selected for validation. Among these genes, COX-2, IFI-6–16 and STS were linked with inflammation, and CSPG2, COMP, CXCR4 and TXNIP were involved in matrix synthesis and cartilage destruction.

The expression profiles of COX-2, IFI-6–16 and CXCR4 showed a significant induction, and STS, CSPG2, COMP and TXNIP were significantly repressed in RASFsn-stimulated chondrocytes compared with NDSFsn-treated controls (Figure 3), thus confirming the gene expression pattern identified by microarray analysis.

RASFsn-stimulated chondrocytes showed a substantial differential expression of genes that were associated with inflammation and cartilage destruction as determined by microarray analysis and real-time RT-PCR. As shown previously, genome-wide microarray analysis of the respective RASF determined a disease-related expression profile of distinct inflammatory mediators [23]. We therefore hypothesized that soluble mediators were secreted from RASF into the supernatant (RASFsn) and induced the catabolic and inflammatory response of chondrocytes after stimulation. Protein analysis of the supernatant of RASF was used to analyze the secretion of soluble mediators by RASF with the use of custom antibody-based cytokine membrane arrays. A proteomic analysis of these supernatants revealed an increased secretion of cytokines/chemokines by RASF (Figure 4); the secretion of IL-6 (spots G3 and G4), CXCL8 (IL-8; spots H3 and H4), monocyte chemoattractant protein-1 (CCL2/MCP-1; spots I3 and I4) and CXCL1–3 (Gro; spots I1 and I2) was increased compared with NDSF. Because cultivation was performed in medium containing serum, the protein content of cultivation medium supplemented with 10% human serum was analyzed as a control.