Detection of differentially expressed genes in synovial fibroblasts by restriction fragment differential display
S. Scaife1,
R. Brown1,
S. Kellie1,4,
A. Filer3,
S. Martin3,
A. M. C. Thomas2,
P. F. Bradfield3,
N. Amft3,
M. Salmon3 and
C. D. Buckley3
1 Yamanouchi Research Institute, Oxford, 2 Royal Orthopaedic Hospital NHS Trust and 3 Department of Rheumatology, Division of Immunity and Infection, University of Birmingham, Birmingham, UK. 4 Present address: School of Molecular and Microbial Sciences and Institute of Molecular Biosciences/CRC for Chronic Inflammatory Diseases, University of Queensland, St Lucia, Brisbane, Qld 4072, Australia.
Correspondence to: C. D. Buckley, Department of Rheumatology, Division of Immunity and Infection, University of Birmingham, Birmingham B15 2TT, UK. E-mail: c.d.buckley{at}bham.ac.uk
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Abstract
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Objective. To identify differentially expressed genes in synovial fibroblasts and examine the effect on gene expression of exposure to TNF-
and IL-1ß.
Methods. Restriction fragment differential display was used to isolate genes using degenerate primers complementary to the lysophosphatidic acid acyl transferase gene family. Differential gene expression was confirmed by reverse transcriptionpolymerase chain reaction and immunohistochemistry using a variety of synovial fibroblasts, including cells from patients with osteoarthritis and self-limiting parvovirus arthritis.
Results. Irrespective of disease process, synovial fibroblasts constitutively produced higher levels of IL-6 and monocyte chemoattractant protein 1 (MCP-1) (CCL2) than skin fibroblasts. Seven genes were differentially expressed in synovial fibroblasts compared with skin fibroblasts. Of these genes, four [tissue factor pathway inhibitor 2 (TFPI2), growth regulatory oncogene ß (GROß), manganese superoxide dismutase (MnSOD) and granulocyte chemotactic protein 2 (GCP-2)] were all found to be constitutively overexpressed in synoviocytes derived from patients with osteoarthritis. These four genes were only weakly expressed in other synovial fibroblasts (rheumatoid and self-limiting parvovirus infection). However, expression in all types of fibroblasts was increased after stimulation with TNF-
and IL-1ß. Three other genes (aggrecan, biglycan and caldesmon) were expressed at higher levels in all types of synovial fibroblasts compared with skin fibroblasts even after stimulation with TNF-
and IL-1.
Conclusions. Seven genes have been identified with differential expression patterns in terms of disease process (osteoarthritis vs rheumatoid arthritis), state of activation (resting vs cytokine activation) and anatomical location (synovium vs skin). Four of these genes, TFPI2, GROß (CXCL2), MnSOD and GCP-2 (CXCL6), were selectively overexpressed in osteoarthritis fibroblasts rather than rheumatoid fibroblasts. While these differences may represent differential behaviour of synovial fibroblasts in in vitro culture, these observations suggest that TFPI2, GROß (CXCL2), MnSOD and GCP-2 (CXCL6) may represent new targets for treatments specifically tailored to osteoarthritis.
KEY WORDS: Rheumatoid arthritis, Inflammation, Differential expression, RFDD, Synoviocytes, Osteoarthritis
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Introduction
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Inflammatory arthritis is characterized by the production of inflammatory cytokines and the accumulation of many different cell types within the inflamed synovium [1,2]. In addition to macrophages and T cells, activated synovial fibroblasts play an important role in driving the persistent inflammation and articular damage seen in rheumatoid arthritis (RA) [2,3]. Changes resulting in the activation of synovial fibroblasts in RA constitute stable long-term alterations that are often maintained in culture even in the absence of proinflammatory cytokines [2]. These changes involve varying degrees of stellate morphology, enhanced growth, increased glucose consumption, altered adherence behaviour and loss of contact inhibition, as well as constitutive overproduction of matrix proteins, metalloproteinases and proinflammatory cytokines such as IL-6 [2]. While this invasive phenotype has not been observed in similarly cultured synoviocytes obtained from synovia from patients with osteoarthritis (OA), it is now becoming clear that in some cases the extent of inflammation observed in OA can match that observed in RA [4].
Synovial fibroblasts are relatively easy to grow in culture and have previously been used to compare gene expression (RA compared with OA) using differential subtraction experiments [5, 6] and an RAP-PCR, cDNA array technique [7]. However, with few exceptions [8], there has been no systematic examination of gene expression profiles in synovial fibroblasts (e.g. RA, OA and self-limiting arthritis) compared with fibroblasts from other tissues. Here we report the use of restriction fragment differential display (RFDD) [9] to look for changes in the expression of genes from the lysophosphatidic acid acyltransferases (LPAAT) family in synovial compared with skin fibroblasts. Members of this family are intrinsic membrane proteins that catalyse the synthesis of phosphatidic acid (PA) from lysoPA and play a key role in inflammation. For example, elevated levels of PA affect tyrosine kinases signalling cascades, vesicle trafficking and cell motility and are involved in regulating the production of proinflammatory cytokines in a number of cell types [10].
We have identified seven differentially expressed genes that, despite not being members of the LPAAT family, contained the shared consensus sequence PEGTRN. Three of these, GROß, biglycan and MnSOD, have previously been identified as being differentially expressed in patients with RA, by differential subtraction [5, 10]. Altered expression of four additional genes, caldesmon, aggrecan-1, GCP-2 and TFPI2, is a novel finding. Four of the genes, GCP-2 (CXCL6), TFPI2, GROß (CXCL2) and MnSOD, were significantly overexpressed in OA-derived fibroblasts compared with other synovial fibroblasts, representing a unique phenotype for OA rather than RA.
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Methods
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Cells and culture conditions
Synovial tissue was obtained at the time of joint replacement from four patients with RA, two with OA and one patient with proven IgM+ self-limiting parvovirus arthritis. Skin fibroblasts were obtained by punch biopsy from the forearm from two patients with mechanical back pain treated with NSAIDs and one healthy volunteer on no treatment. Explant tissue was enzymatically dissociated and cultured through to a maximum of five passages in RPMI 1640 media (Life Technologies), supplemented with 20% fetal calf serum (Life Technologies), 1% penicillin/streptomycin, 1% L-glutamine, 1% sodium pyruvate and 1% non-essential amino acids (Life Technologies) as previously described [8]. Patient details are given in Table 1. Local ethical approval for this study was given (LREC number 5735). Where stated, cells were exposed to 10 ng/ml TNF-
and 1 ng/ml IL-1ß (R & D Systems), in normal growth medium, for 24 h prior to harvesting. In line with current practice, informed consent was sought and obtained from all the patients taking part in this study.
RNA extraction, cDNA synthesis and TaqMan analysis
Total RNA was extracted from the cells by lysis in Trizol solution (Life Technologies), followed by purification on RNEasy columns (Qiagen). DNAse I treatment was applied while the RNA was on the column. One microgram of RNA was reverse-transcribed for use in reverse transcriptionpolymerase chain reaction (RT-PCR) and Taqman (Perkin Elmer) assays for confirmation of differential expression of tissue factor pathway inhibitor 2 (TFPI2). Taqman assays were carried out on an ABI GeneAmp 5700 Sequence Detection System. Results were analysed as detailed in the protocol from the manufacturer. Briefly, values were first normalized to 18S RNA, to account for differences in amounts of starting material. The normalized value for a control sample (in this case untreated skin fibroblasts) was then subtracted from each value, and the result expressed as a fold increase using the formula 2x.
ELISA measurement of IL-6, CCL2 and IL-8 production
Cytokine production was measured in conditioned medium from fibroblasts by enzyme-linked immunosorbent assay (ELISA) as described [8]. Cells were plated at 2 x 105 cells/ml into a 24-well plate. Wells were treated with or without TNF-
and IL-1ß, as described above. After a further 24 h the culture medium was removed and analysed for IL-6, IL-8 and CCL2 using an OptEIA ELISA kits (Pharmingen, San Diego, CA, USA), according to the manufacturer's instructions. The lower detection limits for each ELISA were 15 pg/ml for IL-6, 2 pg/ml for IL-8 and 2 pg/ml for CCL2.
Restriction fragment differential display
Signature motifs or regions of close conservation were first identified from the sequences of the LPAAT gene family and degenerate primers were designed to cover these regions. The functional PEGTRNX motif [12, 13] was used in the reverse orientation: 5'-CCHGARGGVACHMGVAAYCA-3' (degeneracy 648).
Single-stranded cDNA was made by reverse transcription, digested with the restriction endonuclease Taq1, and ligated to an adapter DNA (Display Systems). PCR was carried out on this template, using the degenerate primer and a specific primer complementary to the adapter, incorporating a fluorescent label. The PCR products were then electrophoresed on a 6% polyacrylamide gel (Zaxis) for 1.5 or 3 h at 60 W, in 1 x TrisborateEDTA (TBE) buffer, visualized on a phosphoimager (Typhoon 8600 variable mode imager; Amersham Pharmacia) and the band patterns were compared. The bands of interest were cut from the gel and re-amplified by PCR, using the same primers. After TOPO TA cloning (Invitrogen) of the PCR products, colony PCR and sequencing led to gene identification.
Confirmation of differential expression by RT-PCR
PCR primer sets were designed to amplify unique regions of the genes of interest (see Table 2 for sequences). Amplification was carried out in 25 µl reactions with 12.5 µM of each primer, and PCR mix from ABgene [1.5 mM MgCl2, 1.25 U Taq DNA polymerase, 75 mM TrisHCL pH 8.8, 20 mM (NH4)2SO4, 0.01% Tween 20, 0.2 mM each of dATP, dCTP, dGTP and dTTP]. Template concentrations were normalized to ß-actin levels, by PCR amplification using Clontech ß-actin control primers. Amplification conditions were 94°C for 5 min followed by 30, 32 or 35 cycles of 94°C for 1 min, 55°C for 40 s and 72°C for 1 min.
Immunohistochemistry and microscopy
For live cell staining, 5000 fibroblasts were seeded into eight-well chamber slides (Falcon) and grown to 90100% confluence. The cells were washed in phosphate-buffered saline (PBS), then rabbit anti-human fibronectin antibody (polyclonal, IgG1; Sigma F3648) was added in a 2% (w/v) solution of bovine serum albumin (BSA) in PBS for 30 min at room temperature. After washing, the secondary staining reagent, TRITC-conjugated goat anti-rabbit IgG1 [F(ab)2 fragment; Sigma], was added in a similar way. The cells were then washed and a solution of DAPI (40 ng/ml) was added for 2 min at room temperature to stain cell nuclei. Cells were then washed and visualized. For staining of MnSOD expression, fibroblasts were seeded similarly, but cells were first fixed using acetone before washing and addition of primary (anti-MnSOD mouse monoclonal IgG1; Becton Dickinson 611580) then secondary (fluorescein isothiocyanate-conjugated goat anti-mouse pan IgG; Southern Biotechnology Associates 1031-02) antibodies. The cells were washed and treated with DAPI as before, then mounted in 1% (w/v) P-phenylene diamine, 90% (v/v) glycerol.
Fibroblasts were imaged by differential interference contrast and epifluorescence microscopy using a Zeiss Axiovert 200 microscope. Images were captured and merged using a Hamamatsu C4742-95 camera and Simple PCI software (Digital Pixel, Brighton, UK).
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Results
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Synovial fibroblasts produce high levels of IL-6 compared with skin fibroblasts irrespective of disease process
It has been well documented that rheumatoid fibroblasts constitutively produce large quantities of a variety of inflammatory cytokines and chemokines, such as IL-6, IL-8 and CXCL2 [14]. However, it is not clear whether this is a feature intrinsic to rheumatoid fibroblasts or more general to synovial fibroblasts irrespective of disease process. In order to examine this and to help choose representative cell lines for further study, we examined the morphology, production of fibronectin and secretion of IL-6, IL-8 and CCL2 from fibroblasts isolated from RA (four lines), OA (two lines) and self-limiting arthritis (one line) as well as skin (three lines) (Table 1). As described previously [8, 15] rheumatoid synovial fibroblasts exhibited a more heterogeneous stellate morphology compared with skin fibroblasts (Fig. 1A). Interestingly, the two fibroblasts from osteoarthritic synovia were more similar to rheumatoid fibroblasts, whereas the self-limiting fibroblast cell line was more similar to skin, with a more homogeneous spindle-shaped morphology. In general, skin fibroblasts were larger with larger nuclei. The production of fibronectin (measured by immunofluorescence on live unfixed cells) was variable between cell lines but in general was highest in the rheumatoid fibroblasts.


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FIG. 1. Morphology and cytokine production from synovial and skin fibroblasts. (A) Fibroblasts were examined by immunohistochemistry as described in Methods. Nuclei are stained blue and fibronectin red (this figure may be viewed in colour as supplementary data at Rheumatology Online). (B) Conditioned medium from the fibroblasts shown in (A) was analysed for IL-6, MCP-1 (CCL2) and IL-8 by ELISA. The data are shown as a box plot extending from the 25th to the 75th percentile with a line at the median. The whiskers extend above and below the box to show the highest and lowest values. Data were pooled from all fibroblasts representing each condition, with a minimum of three data points for each fibroblast. This figure may be viewed in colour as supplementary data at Rheumatology Online.
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The production patterns of IL-6, IL-8 and CCL2 by the different fibroblasts confirmed our previous findings of high levels of IL-6 and CCL2 in rheumatoid compared with skin fibroblasts [8]. Interestingly constitutive IL-6 and CCL2 production was also high in fibroblasts from OA and self-limiting arthritis, suggesting that the production of these cytokines is a generic property of all synovial fibroblasts irrespective of disease state. As predicted from our previous studies [8], after 24 h stimulation with IL-1ß and TNF-
the expression of IL-6 and CCL2 by all synovial fibroblasts remained higher than expression by skin fibroblasts (data not shown). Although basal IL-8 production was low in all fibroblasts, after stimulation with IL-1ß and TNF-
synovial fibroblasts consistently expressed higher levels of IL-8 than skin fibroblasts, confirming our previous findings [8].
Identification of seven differentially expressed genes in synovial compared with skin fibroblasts by RFDD
Having demonstrated clear differences in phenotype between rheumatoid synovial fibroblasts and skin fibroblasts, we next looked for differences in mRNA expression patterns by RFDD, using both untreated and cytokine-stimulated cells. We specifically wanted to isolate new members of the LPAAT gene family, so primers were used to amplify potential conserved regions (see Methods). We chose RA-1 as a representative rheumatoid fibroblast cell line to compare against the skin fibroblast cell line SK-1. An example of a typical RFDD gel is shown in Fig. 2. Several bands showing distinct expression differences were selected for further analysis (red boxes; see figure in colour as supplementary data at Rheumatology Online).

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FIG. 2. Differential expression by RFDD. The differentially expressed genes are marked with red boxes. They are (smallest first): GROß, caldesmon, MnSOD, biglycan, GCP-2 and aggrecan-1. TFPI2 was analysed on a separate gel (not shown here), which was expressed on 24-h stimulated rheumatoid fibroblasts but not skin fibroblasts. This figure may be viewed in colour as supplementary data at Rheumatology Online.
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DNA extraction, re-amplification, cloning and nucleotide sequencing of the differentially expressed bands revealed seven distinct genes. Comparison of the sequences with the GenBank database showed 100% homology with previously identified human genes. These were: tissue factor pathway inhibitor 2 (TFPI2), growth regulatory oncogene ß (GROß), caldesmon, manganese superoxide dismutase (MnSOD), biglycan, granulocyte chemotactic protein 2 (GCP-2) and aggrecan 1. These results are summarized in Table 3.
All of the differentially expressed genes identified using the LPAAT degenerate primers contained at least some of the amino acid PEGTR motif on which the primer sequence was based. Therefore, while this technique worked in recognizing specific motifs, the motif used here was not specific to LPAATs as no members of the LPAAT family were identified in this study.
Confirmation of differential expression by RT-PCR and immunohistochemistry
To confirm that the expression of these seven genes was differential between rheumatoid and skin fibroblasts and to extend our observations more generally, we used RT-PCR to assess the approximate expression level of each gene in our panel of fibroblasts. The PCR conditions were optimized so that amplification was semiquantitative and the quantity of starting cDNA was normalized for ß-actin. Figure 3 shows that these genes, identified by RFDD, show marked differential expression, both between tissue samples and with cytokine treatment.
TFPI2 was weakly expressed by all untreated skin fibroblasts, rheumatoid and parvovirus synoviocytes, but was constitutively expressed by both samples of OA synoviocytes. Treatment with TNF-
and IL-1ß increased expression of TFPI2 in all samples except for the OA synoviocytes. In contrast, aggrecan-1 expression was highest in rheumatoid fibroblasts, TNF-
and IL-1ß stimulation having little effect. Caldesmon and biglycan were expressed by all fibroblasts with no obvious effect of cytokine treatment.
The most striking finding was the enhanced expression of GCP-2, GROß, MnSOD and TFPI2 in the untreated OA fibroblasts compared with other untreated fibroblasts. This increase was confirmed at the mRNA level by Taqman analysis of TFPI2 (Fig. 4A), as well as at the protein level for MnSOD (Fig. 4B). However, stimulation with IL-1ß and TNF-
led to an increase in TFPI-1 mRNA and MnSOD in most of the fibroblasts, although this was variable between fibroblasts.
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Discussion
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In this study we have demonstrated two specific differential gene expression patterns in fibroblasts derived from synovium (RA, OA, parvovirus infection) and skin. First, there are genes which are more highly expressed in synovial fibroblasts compared with skin fibroblasts (i.e. in a site-specific manner). Secondly, there are genes which are differentially expressed between OA and RA synoviocytes and show a different response to cytokine treatment (i.e. disease-specific expression).
Using a panel of synovial fibroblasts, we found that, irrespective of disease process, synovial fibroblasts produce high levels of the cytokine IL-6 and the chemokine CCL2, although there was a trend towards higher levels of expression in RA and OA fibroblasts compared with fibroblasts from self-limiting arthritis. This suggests that constitutive IL-6 and CCL2 production from synovial fibroblasts represents a common synovial response to injury. Our findings are in keeping with the emerging view that OA can be considered a localized inflammatory disease compared with RA, which is a more systemic inflammatory syndrome. In line with this, a number of studies have shown that in patients with OA the extent of inflammation and particularly inflammatory cell infiltrate can reach that observed in RA [1618]. Of note, both OA synovial fibroblast samples in this study were derived from patients with severe OA with histological evidence of heavy inflammatory infiltrates (data not shown).
The major cartilage proteoglycans are aggrecan, biglycan and decorin [19]. Biglycan is a small cellular or pericellular matrix proteoglycan, containing 11 tandem repeats of a leucine-rich 24 amino acid sequence [20]. It has been reported as being more abundant in proliferating cartilage, as would be expected in arthritic joints.
TNF-
has previously been found to induce chondrocytes to degrade proteoglycans in their surrounding matrix through limited proteolysis and to inhibit the synthesis of new proteoglycans [19]. Incubation of chondrocytes with low doses of TNF-
and IFN-
caused a decrease in the levels of mRNA for aggrecan (45%) and biglycan (48%) [14]. Similarly, Demoor-Fossard [21] reported that IL-1ß down-regulated the expression of aggrecan in chondrocytes. In our studies, stimulation with TNF and IL-1 did not affect aggrecan or biglycan mRNA expression in synoviocytes, but did seem to down-regulate aggrecan expression in skin-derived fibroblasts.
Caldesmon is a high molecular weight protein that binds actin and calciumcalmodulin [22, 23]. It was initially purified as a component of smooth muscle thin filaments and was found to be able to inhibit the actin-tropomyosin ATPase. Non-muscle caldesmons regulate cell motility and cytoskeletal organization [24] and have also been implicated in leucocyte activation [25]. Although this gene appeared to be differentially expressed between rheumatoid and skin fibroblasts in the RFDD analysis, we did not observe any consistent differences in RT-PCR studies in a wider panel of fibroblasts.
In contrast, four other genes (GROß, GCP-2, MnSOD and TFPI2) were all constitutively expressed in OA synoviocytes, showing no further increase in expression upon cytokine stimulation. These genes were expressed at very low levels in rheumatoid, parvovirus and skin fibroblasts but were up-regulated upon cytokine stimulation, suggesting that they can be regulated during the inflammatory response. Furthermore, we confirmed that the differential expression between resting OA and RA fibroblasts we observed occurred for TFPI2 using Taqman PCR and at the protein level for MnSOD using immunohistochemical studies of resting and activated fibroblasts.
In agreement with our results, constitutive expression of GROß (CXCL2) in OA synoviocytes has been reported and treatment with anti-GROß antibodies has been shown to delay the onset and reduce the severity of collagen-induced arthritis [26, 27]. GRO
, ß and
are known to induce chemotaxis, changes in shape, a transient rise in cytosolic Ca2+, granule exocytosis and respiratory burst in neutrophils, thus acting as mediators in inflammation [28, 29]. Therefore, up-regulation of GROß is likely to exacerbate the inflammatory response.
GCP-2 (CXCL6) is a member of the ELR-expressing CXC subfamily of chemokines, and acts as a potent chemoattractant of neutrophils in the course of acute inflammation [30, 31]. It binds and activates two receptors, CXCR1 and CXCR2, which are expressed on neutrophils. GCP-2 is the only ELR+ CXC chemokine, except for IL-8, that is an effective ligand for CXCR1 in addition to CXCR2. Intradermal injection of GCP-2 or GRO in normal rabbit skin provokes prominent granulocyte infiltration. Up-regulation of GCP-2 by cytokines has also been previously reported [30].
Superoxide dismutases (SOD), such as MnSOD, are metalloproteinases that convert oxygen radicals to hydrogen peroxide (H2O2). TNF-
has been shown to increase MnSOD expression in a variety of human cell lines. A number of potentially toxic elements are released at the site of inflammation, including oxygen radicals. Direct injection of superoxide dismutase into inflamed joints has been reported to partially relieve the symptoms of RA; this may be due either to removal of superoxide radicals or to a non-specific protein effect. Nivsarkar [32] found that in RA patients superoxide dismutase activity was significantly lower than in controls, but levels of circulating SOD were improved after NSAID therapy.
TFPI2 is a Kunitz-type serine proteinase inhibitor, isolated by virtue of its sequence homology to TFPI1 [33]. It is generally associated with the extracellular matrix (ECM) and is an inhibitor of trypsin, plasmin, plasma kallikrein, factor XIa, chymotrypsin, factor VIIa-tissue factor and cathepsin G [34]. TNF-
stimulation of HUVEC and human vascular smooth muscle cells has previously been shown to increase TFPI2 expression (and release) in a time and dose-dependent manner [34, 35]. TFPI2 may serve a protective function in inflammation, by inhibiting matrix metalloproteinase activation.
As a technique, RFDD compares favourably with other methods for the detection of differential gene expression, such as differential subtraction, hybridization and genome-wide microarray analysis. Differential subtraction and hybridization techniques are labour-intensive and require large quantities of RNA, which are often not easily obtained from diseased samples. In addition, these techniques cannot target specific gene sequences. Although microarray analysis using targeted arrays can identify specific gene families, these often require large amounts of sample, and will not detect novel members of specific gene families. RFDD does not suffer from these limitations and also allows fingerprints from several mRNA populations to be easily compared side-by-side on one gel [36]. Our work demonstrates the successful use of RFDD in identifying differentially expressed genes that contain a common motif. Although no differentially expressed members of the LPAAT family were identified in this study, six of the seven genes identified contained all or part of the LPAAT motif, amino acids PEGTRNX. TFPI2 did not show any part of the PTPase signature motif, and was presumably detected due to the high degeneracy of the primers used.
This paper reports the successful use of the RFDD technique to isolate seven genes which are differentially regulated in synovial and skin fibroblasts, and are further affected by TNF-
and IL-1ß. The genes identified here allow a clear distinction to be made between fibroblasts derived from different anatomical sites (synovial vs skin) as well as different synovial diseases (OA vs RA). Of particular interest is the finding that proinflammatory genes such as GROß (CXCL2) and GCP-2 (CXCL6), implicated in the recruitment of leucocytes to sites of inflammation, are constitutively expressed in resting OA but not rheumatoid fibroblasts, supporting the idea that in some circumstances OA can be considered an inflammatory disease [4]. While some of the differences we have observed between RA- and OA-derived fibroblasts may represent differences due to in vitro culture, we propose that these differences might facilitate a clearer understanding of those features of inflammation that are a common response to synovial inflammation (i.e. generic to OA and RA) from those that are more disease-specific and represent potential new targets for anti-inflammatory drugs tailored to OA.
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Acknowledgments
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We would like to thank Dr Craig Southern for advice and helpful discussions about the manuscript. This work was partially funded from grants from the ARC and MRC.
C. D. Buckley received funding for a PhD studentship from Yamanouchi Research Institute. The other authors have declared no conflicts of interest.
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References
|
---|
- Buckley CD. Science, medicine and the future: treatment of rheumatoid arthritis. BMJ 1997;315:2368.[Free Full Text]
- Pap T, Muller-Ladner U, Gay RE, Gay S. Role of synovial fibroblasts in the pathogenesis of rheumatoid arthritis. Arthritis Res 2000;2:3617.[CrossRef][ISI][Medline]
- Buckley CD, Pilling D, Lord JM, Akbar AN, Scheel-Toellner D, Salmon M. Fibroblasts regulate the switch from acute resolving to chronic persistent inflammation. Trends Immunol 2001;22:199204.[CrossRef][ISI][Medline]
- Pelletier J-P, Martel-Pelletier J, Abramson SB. Osteoarthritis, an inflammatory disease? Arthritis Rheum 2001;44:123747.[CrossRef][ISI][Medline]
- Seki T, Selby J, Haupl T, Winchester R. Use of a differential subtraction method to identify genes that characterize the phenotype of cultured rheumatoid arthritis synoviocytes. Arthritis Rheum 1998;41:135664.[CrossRef][ISI][Medline]
- Justen H-P, Grunewald E, Totzke G et al. Differential gene expression in synovium of rheumatoid arthritis and osteoarthritis. Mol Cell Biol Res Commun 2000;3:16572.[CrossRef][Medline]
- Neaumann E, Kullmann F, Judex M et al. Identification of differentially expressed genes in rheumatoid arthritis by a combination of complementary DNA array and RNA arbitrarily primed-polymerase chain reaction. Arthritis Rheum 2002;46:5263.[CrossRef][ISI][Medline]
- Parsonage G, Falciani F, Burman A et al. Global gene expression profiles in fibroblasts from synovial, skin and lymphoid tissue reveals distinct cytokine and chemokine expression patterns. Thromb Haemost 2003;90:68897.[ISI][Medline]
- Fischer A, Saedler H, Theissen G. Restriction fragment length polymorphism-coupled domain-directed differential display: a highly efficient technique for expression analysis of multigene families. Proc Natl Acad Sci USA 1995;92:53315.[Abstract]
- West J, Tompkins CK, Balantac N et al. Cloning and expression of two human lysophosphatidic acid acyltransferase cDNAs that enhance cytokine-induced signaling responses in cells. DNA Cell Biol 1997;16:691701.[ISI][Medline]
- Stuhlmuller B, Ungethum U, Acholze S et al. Identification of known and novel genes in activated monocytes from patients with rheumatoid arthritis. Arthritis Rheum 2000;43:77590.[CrossRef][ISI][Medline]
- Lewin TM, Wang P, Coleman RA. Analysis of amino acid motifs diagnostic for the sn-glycerol-3-phosphate acyltransferase reaction. Biochemistry 1999;38:576471.[CrossRef][ISI][Medline]
- Stamps AC, Elmore MA, Hill ME, Kelly K, Makda AA, Finnen MJ. A human cDNA sequence with homology to non-mammalian lysophosphatidic acid acyltransferases. Biochem J 1997;326:45561.[ISI][Medline]
- Bucala R, Ritchlin C, Winchester R, Cerami A. Constitutive production of inflammatory and mitogenic cytokines by rheumatoid synovial fibroblasts. J Exp Med 1991;173:56974.[Abstract]
- Firestein GS. Invasive fibroblast-like synoviocytes in rheumatoid arthritis. Passive responders or transformed aggressors? Arthritis Rheum 1996;39:178190.[ISI][Medline]
- Haraoui B, Pelletier J-P, Cloutier J-M et al. Synovial membrane histology and immunopathology in rheumatoid arthritis and osteoarthritis: in vivo effects of antirheumatic drugs Arthritis Rheum 1991;34:15363.[ISI][Medline]
- Farahat MN, Yanni G, Poston R et al. Cytokine expression in synovial membranes of patients with rheumatoid arthritis and osteoarthritis. Ann Rheum Dis 1993;52:8705.[Abstract]
- Smith MD, Triantafillou S, Parker A et al. Synovial membrane inflammation and cytokine production in patients with early osteoarthritis. J Rheumatol 1997;24:36571.[ISI][Medline]
- Dodge GR, Diaz A, Sanz-Rodriguez C, Reginato AM, Jimenez SA. Effects of Interferon-
and tumor necrosis factor
on the expression of the genes encoding aggrecan, biglycan, and decorin core proteins in cultured human chondrocytes. Arthritis Rheum 1998;41:27483.[CrossRef][ISI][Medline]
- Fisher LW, Heegaard A-M, Vetter U et al. Human biglycan gene. Putative promoter, intron-exon junctions, and chromosomal localization. J Biol Chem 1991;299:143717.
- Demoor-Fossard M, Redini F, Boittin M, Pujol J-P. Expression of decorin and biglycan by rabbit articular chondrocytes. Effects of cytokines and phenotypic modulation. Biochim Biophys Acta 1998;1398:17991.[ISI][Medline]
- Huber AJ. Caldesmon. Int J Biochem Cell Biol 1997;29:104751.[CrossRef][ISI][Medline]
- Matsumura F, Yamashiro S. Caldesmon. Curr Opin Cell Biol 1993; 5:706.[Medline]
- Helfman DM, Levy ER, Berthier C et al. Caldesmon inhibits nonmuscle cell contractility and interferes with the formation of focal adhesions. Mol Biol Cell 1999;10:3097112.[Abstract/Free Full Text]
- Arias MP, Pacaud M. Macrophage caldesmon is an actin bundling protein. Biochemistry 2001;40:1297482.[CrossRef][ISI][Medline]
- Haskill S, Peace A, Morris J et al. Identification of three related human GRO genes encoding cytokine functions. Proc Natl Acad Sci USA 1990;87:77326.[Abstract]
- Hogan M, Sherry B, Ritchlin C et al. Differential expression of the small inducible cytokines GRO
and GROß by synovial fibroblasts in chronic arthritis: possible role in growth regulation. Cytokine 1994;6:619.[CrossRef][ISI][Medline]
- Geiser T, Dewald B, Ehrengruber MU, Clark-Lewis I, Baggiolini M. The interleukin-8-related chemotactic cytokines GRO
, GROß, and GRO
activate human neutrophil and basophil leukocytes. J Biol Chem 1993;268:1541924.[Abstract/Free Full Text]
- Iida N, Grotendorst GR. Cloning and sequencing of a new GRO transcript from activated human monocytes: expression in leukocytes and wound tissue. Mol Cell Biol 1990;10:55969.[ISI][Medline]
- Feniger-Barish R, Belkin D, Zasiaver A et al. GCP-2-induced internalization of IL-8 receptors: hierarchical relationships between GCP-2 and other ELR+-CXC chemokines and mechanisms regulating CXCR2 internalization and recycling. Blood 2000;95:15519.[Abstract/Free Full Text]
- Proost P, de Wolf-Peeters C, Conings R, Opdenakker G, Billiau A, Van Damme J. Identification of a novel granulocyte chemotactic protein (GCP-2) from human tumor cells. J Immunol 1993;150:100010.[Abstract/Free Full Text]
- Nivsarkar M. Improvement in circulating superoxide dismutase levels: role of nonsteroidal anti-inflammatory drugs in rheumatoid arthritis. Biochem Biophys Res Commun 2000;270:7146.[CrossRef][ISI][Medline]
- Sprecher CA, Kisiel W, Mathewes S, Foster DC. Molecular cloning, expression, and partial characterization of a second human tissue-factor-pathway inhibitor. Proc Natl Acad Sci USA 1994;91:33537.[Abstract]
- Iino M, Foster DC, Kisiel W. Quantification and characterization of human endothelial cell-derived tissue factor pathway inhibitor-2. Arterioscler Thromb Vasc Biol 1998;18:406.[Abstract/Free Full Text]
- Herman MP, Sukhova GK, Kisiel W et al. Tissue factor pathway inhibitor-2 is a novel inhibitor of matrix metalloproteinases with implications for atherosclerosis. J Clin Invest 2001;107:111726.[Abstract/Free Full Text]
- Ivanova NB, Belyavsky AV. Identification of differentially expressed genes by restriction endonuclease-based gene expression fingerprinting. Nucleic Acids Res 1995;23:29548.[Abstract]
Submitted 20 February 2003;
revised version accepted 29 June 2004.