MLN51and GM-CSF involvement in the proliferation of fibroblast-like synoviocytes in the pathogenesis of rheumatoid arthritis
© Jang et al.; licensee BioMed Central Ltd. 2006
Received: 15 May 2006
Accepted: 14 November 2006
Published: 14 November 2006
Rheumatoid arthritis (RA) is an inflammatory autoimmune disease of unclear etiology. This study was conducted to identify critical factors involved in the synovial hyperplasia in RA pathology. We applied cDNA microarray analysis to profile the gene expressions of RA fibroblast-like synoviocytes (FLSs) from patients with RA. We found that the MLN51 (metastatic lymph node 51) gene, identified in breast cancer, is remarkably upregulated in the hyperactive RA FLSs. However, growth-retarded RA FLSs passaged in vitro expressed small quantities of MLN51. MLN51 expression was significantly enhanced in the FLSs when the growth-retarded FLSs were treated with granulocyte – macrophage colony-stimulating factor (GM-CSF) or synovial fluid (SF). Anti-GM-CSF neutralizing antibody blocked the MLN51 expression even though the FLSs were cultured in the presence of SF. In contrast, GM-CSF in SFs existed at a significant level in the patients with RA (n = 6), in comparison with the other inflammatory cytokines, IL-1β and TNF-α. Most RA FLSs at passage 10 or more recovered from their growth retardation when cultured in the presence of SF. The SF-mediated growth recovery was markedly impaired by anti-GM-CSF antibody. Growth-retarded RA FLSs recovered their proliferative capacity after treatment with GM-CSF in a dose-dependent manner. However, MLN51 knock-down by siRNA completely blocked the GM-CSF/SF-mediated proliferation of RA FLSs. Taken together, our results imply that MLN51, induced by GM-CSF, is important in the proliferation of RA FLSs in the pathogenesis of RA.
Synovial tissue from healthy individuals consists of a single layer of synovial cells without infiltration of inflammatory cells. In rheumatoid synovial tissue, lymphocytes and macrophages are recruited and activated, and these activated macrophages release high concentrations of inflammatory cytokines. In response to these cytokines, synovial fibroblasts proliferate vigorously and form villous hyperplastic synovial tissues. These fibroblasts secrete inflammatory mediators, which further attract inflammatory cells and stimulate the growth of the synovial fibroblasts and vascular endothelial cells . These activated macrophages and fibroblasts produce tissue-degrading proteinases . Thus, invasive hyperplastic synovial tissue, termed pannus, is directly responsible for the structural and functional damage to the affected joints. Therapeutic intervention against rheumatoid arthritis (RA) could aim at any one of the aforementioned steps, but the driving mechanisms underlying this process are largely unknown. Impaired regulation of apoptosis has been associated with RA [3–5]; however, apoptosis of synovial cells has been identified in rheumatoid synovium [6, 7], which suggests that synovial tissue hyperplasia may be a result of cell proliferation rather than apoptotic cell death [8–10].
This study was initiated to address the molecular characterization of fibroblast-like synoviocyte (FLS) hyperproliferation in RA pathogenesis. We used cDNA microarray technology to identify genes related to the proliferation of RA FLSs. We found that the expression of the MLN51 (metastatic lymph node 51) gene was markedly enhanced in RA FLSs when cultured in the presence of the RA synovial fluid (SF). MLN51 was first identified in breast cancer cells, and the same investigators subsequently reported that MLN51 associates with exon junction complexes in the cell nucleus and remains stably associated with mRNA in the cytoplasm [11, 12]. Recently, the interactions of MLN51 with other exon junction complex components, a clamping mechanism on mRNAs, and some additional biological functions of MLN51 in the exon junction complex core have been identified and addressed [13–15].
Our series of experimental results have demonstrated that MLN51 is important in the hyperproliferation of RA FLSs in the presence of granulocyte – macrophage colony-stimulating factor (GM-CSF) in SF. These results strongly suggest that the MLN51 gene would be an ideal target for the development of new RA therapeutics.
Materials and methods
Isolation and establishment of RA FLSs from patients with RA
FLS cells (designated RA s-2, 2–6, 2–14, 2–18, 2–36 and 2–38) were prepared from synovectomized tissue of six patients with RA undergoing joint replacement surgery at the Kangnam St Mary Hospital, Catholic University of Korea, Seoul, Korea. Institutional Board Approval (IRB) and informed patient consent were obtained for each enrolled participant. The mean age of the patients was 43.7 years and their disease duration was greater than 24 months. The patients had visible joint erosions by radiography of the hand, and all satisfied the diagnostic criteria of the American College of Rheumatology (formerly the American Rheumatism Association) for the classification of RA . RA FLSs 2–14, 2–18, 2–36 and 2–38 among the above FLSs could be subjected to Western blot analysis because their sample amounts were sufficient. RA FLSs were prepared as described previously [17–19]. In brief, synovial tissues were minced into pieces 2 to 3 mm in size and treated for 4 hours with 4 mg/ml type 1 collagenase (Worthington Biochemicals, Freehold, NJ, USA) in DMEM at 37°C in 5% CO2. Dissociated cells were centrifuged at 500 g for 10 minutes and were resuspended in DMEM supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. Suspended cells were plated in 75 cm2 culture flasks and cultured at 37°C in 5% CO2. Medium was replaced every 3 days, and once the primary culture had reached confluence, cells were split weekly. Cells at passages 5 to 8 were morphologically homogenous and had the appearance of FLSs with typical bipolar configuration under inverse microscopy (less than 2.5% CD14+, less than 1% CD3+ and less than 1% CD19+ in flow cytometry analysis) . Osteoarthritis (OA) FLSs (designated OA 2–43, 2–46 and 2–47) were used as controls and were prepared from the synovial tissues of three confirmed and enrolled patients with OA. Synovial fluid samples were obtained from the knee joints of different six patients with active RA.
Generation of mouse bone marrow-derived dendritic cells
Immature bone marrow-derived dendritic cells (BmDCs) were generated from bone marrow precursor cells of DBA/1J mice (obtained from the Jackson Laboratory, Bar Harbor, ME, USA) as described previously . In brief, bone marrow cells were harvested from the femurs and tibias of mice and plated in RPMI-1640 medium supplemented with 10% FBS, 50 μM 2-mercaptoethanol, and high-dose (200 U/ml) murine GM-CSF (Endogen, Inc., Cambridge, MA, USA). The medium was changed every other day. Seven days later, non-adherent cells (immature DCs) were harvested by gentle washing with warm PBS. For DC maturation, cells were stimulated for 24 hours with TNF-α (500 U/ml; Endogen) or with lipopolysaccharide (E. coli, 0127:B8; 1 μg/ml; Sigma-Aldrich, St Louis, MO, USA) together with anti-CD40 (clone 3/23 or HM40, 5 μg/ml; BD Pharmingen, San Jose, CA, USA). The purity and maturation status of DCs were analyzed by a flow cytometer (FACSCalibur; BD Biosciences, San Jose, CA, USA) with the use of fluorescein isothiocyanate-conjugated CD44, CD80, CD86, CD205 and MHC II mAbs or phycoerythrin-conjugated CD11c, CD40 and ICOSL mAbs (BD Pharmingen, San Diego, CA, USA). Data were analyzed with Cell Quest Software.
DC cell line
BC-1 cells, from the DC cell line generated from BALB/c mouse spleen [21, 22], were kindly provided by Dr Onoe (Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan). BC-1 cells were cultured and expanded in Iscove's modified Dulbecco's medium containing 10% FCS, 30% NIH/3T3 culture supernatant, and 10 ng/ml mouse recombinant GM-CSF. Cultured cells exhibit an immature DC phenotype.
cDNA microarray analysis of rheumatoid arthritis fibroblast-like synoviocytes
Two types of immunologic cDNA microarray chip, namely HI380 and MI380 (Creagene Inc., Seoul, Korea) described previously , were used in this study (MI380 microarray data were not shown in the present report. Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA) and purified by using the RNeasy total RNA isolation kit (Qiagen, Valencia, CA, USA) in accordance with the manufacturer's instructions. The gene expression profile of human RAFLSs and mouse BmDCs were analyzed with the HI380 and MI380 microarray chips, consisting of 384 human and mouse cDNA clones, respectively. Total RNA (20 μg) was reverse-transcribed in the presence of Cy-3-conjugated or Cy-5-conjugated dUTP (Amersham Pharmacia Biotech, Piscataway, NJ, USA), using SuperScript II and oligo(dT)18 primer (Invitrogen) in a reaction volume of 20 μl in accordance with the method suggested by the manufacturer. After the labeling reaction for 1 hour at 42°C, unincorporated florescent nucleotides were cleaned up with a Microcon YM-30 column (Millipore, Bedford, MA, USA). The Cy-3-labeled and Cy-5-labeled cDNA probes were mixed together and hybridized to a microarray slide. After incubation overnight at 65°C, the slide was washed twice with 2 × SSC containing 0.1% SDS for 5 minutes at 42°C, once with 0.1 × SSC containing 0.1% SDS for 10 minutes at room temperature, and finally with 0.1 × SSC for 1 minute at room temperature. Slides were dried by centrifugation at 650 r.p.m. for 5 minutes. Hybridization images on the slide were scanned with a Scanarray lite (Packard Bioscience, Boston, MA, USA) and analyzed with GenePix Pro3.0 software (Axon Instruments, Union City, CA, USA). Three separate and independent experiments were performed and the ratio of Cy-3 and Cy-5 signal intensities was calculated for each spot. These ratios were log2-transformed and normalized by subtracting the average of log2(Cy-3/Cy-5) values for internal control genes by using Excel (Office 2003; Microsoft Corp.) . For each gene, the mean values were then calculated and a twofold difference was applied to select upregulated or downregulated genes in RA/OA FLSs or immature DC/bone marrow progenitors.
To confirm the upregulation or downregulation of the selected gene (MLN51) on the microarray analysis and the expression of MLN51 after siRNA transfection, total RNAs were extracted from RA FLSs with Trizol reagent (Invitrogen) and purified with an RNeasy total RNA isolation kit (Qiagen) in accordance with the manufacturer's instructions. Total RNA (1 μg) was mixed with 50 μM oligo(dT)20, and 10 mM dNTP mixture, heated at 65°C for 5 minutes, and placed on ice for at least 1 minute. Then 10 × RT buffer (25 mM MgCl2, 0.1 M dithiothreitol, RNaseOUT™ (40 U)) and 1 μl of SuperScript™ III reverse transcriptase (200 U/μl; Invitrogen) were added, and the mixture was incubated at 42°C for 1 hour. The reaction was terminated by incubation at 75°C for 5 minutes followed by chilling on ice. The PCR was performed with the cDNA as template and certain gene specific-primers.
The following primers were used in this study: hMLN51 forward, 5'-AAGACACCGAGGACGAGGAATC-3', hMLN51 reverse, 5'-CCTTCCATAGCTTTCGCTGACG-3', product size 600 base pairs; mMLN51 forward, 5'-TCCCTGCCCTGCCCTGACTTTA-3', mMLN51 reverse, 5'-CCTCGCGTGCTGTGGGAACTCT-3', product size 800 bp; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward, 5'-CCACAGTCCATGCCATCAC-3', GAPDH reverse, 5'-TCCACCACCCTGTTGCTGTA-3', product size 500 bp. The initial cDNA content in each sample was normalized with the amount of GAPDH. Amplification reactions were performed in a 20 μl volume with 5 or 10 ng of each cDNA on a Perkin-Elmer DNA thermocycler 9600 Prism for 35 cycles. The PCR reactions were separated on 1.2% agarose gels and stained with ethidium bromide.
Measurement of cytokine levels in rheumatoid arthritis synovial fluid
IL-1β and TNF-α were measured in the SFs with the Human Cytometric Bead Array (BD Pharmingen, San Diego), and GM-CSF was measured with the human ELISA kit (Endogen) in accordance with the manufacturer's instructions.
Western blot analysis
RA FLS samples were lysed in boiled buffer containing 1% SDS. Each sample, containing a normalized amount of total protein (about 30 μg of protein), was separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. This was then immersed in blocking buffer (5% skimmed milk and 0.1% Tween 20 in PBS, pH 7.4) for 1 hour at room temperature and incubated with anti-hMLN51 rabbit serum (1:1,000 dilution) and anti-GAPDH (1:5,000 dilution) or anti-α-tubulin (1:5,000 dilution) in blocking buffer overnight at 4°C. Anti-hMLN51 serum was obtained from rabbits immunized with recombinant hMLN51 protein. After the incubation, the membrane was probed with horseradish peroxidase-labeled anti-rabbit IgG antibody (1:5,000 dilution) in PBS (containing of 0.05% Tween 20 and 5% skimmed milk powder) for 30 minutes at room temperature. The proteins in the membrane were detected by enhanced chemiluminescence (Amersham, Little Chalfont, Bucks., UK) and bands were detected by autoradiography with X-ray film (Fujifilm).
Treatment of rheumatoid arthritis fibroblast-like synoviocytes with synovial fluid, cytokine or neutralizing antibodies
RA FLSs were cultured in 12-well plates in high-glucose DMEM supplemented with 10% FBS at 37°C in a 5% CO2 humidified incubator. For SF and cytokine treatments, RA FLSs were treated with SFs serially diluted in culture medium. Inflammatory cytokines (IL-1β and TNF-α; 100 ng/ml of each) and the growth factor (GM-CSF; 10 or 100 ng/ml) were obtained from PeproTech (Rocky Hill, NJ, USA) or BD Pharmingen (San Diego). Neutralizing monoclonal antibodies against GM-CSF (BVD2-23B6, IgG2a; 300 ng/ml), IL-1β (AS10, IgG1; 500 ng/ml) and TNF-α (MAb1, IgG1; 2 μg/ml) were purchased from BD Pharmingen (San Diego). RA FLSs were preincubated with these neutralizing antibodies for 1 hour. The trypan blue exclusion method was used for the evaluation of cell proliferation during all experiments.
siRNA synthesis and transfection
siRNA synthesis was performed with the Silencer™ siRNA Cocktail Kit (RNase III; Ambion Inc., Austin, Texas, USA). The siRNA sequence was used for targeted silencing of human MLN51 (GenBank accession number NM007359) and mouse MLN51 (GenBank accession number AJ292072). The oligonucleotides used for the dsRNA synthesis were, in hMLN51, 5'-TAATACGACTCACTATAGGGTACTCGTAAGATGGCGGACCGG-3' and 5'-TAATACGACTCACTATAGGGTCCGTCCCCACTTTGCCTC-3', and in mMLN51, 5'-TAATACGACTCACTATAGGGTACTCGTAAGATGGCGGACCGG-3' and 5'-TAATACGACTCA CTATAGGGTACTCTGCCTCTCCCCAGTCAC-3'. The siRNA sequences were selected in size ranging from 228 to 686 bp, as described previously [25, 26]. The siRNA synthesis was performed in accordance with the manufacturer's protocol. Non-silencing or negative control siRNA (Silencer Negative Control no. 2 siRNA; Ambion Inc.) is an irrelevant siRNA with random nucleotides and no known specificity. RA FLSs (RA 2–14, at passage 5; 104 per well) and BC-1 cells (104 per well) were seeded in 24-well plates in DMEM supplemented with 10% FBS and Iscove's modified Dulbecco's medium (containing 10% FCS), respectively. The cells were transfected with the siRNA (4 μg) on the next day, with the GenePORTER 2 Transfection reagent™ (Gene Therapy Systems, San Diego, CA, USA) in accordance with the manufacturer's protocol. At 24 hours after transfection, fresh culture medium was added to the medium. Cells were harvested every day and counted. Total RNA extracted from the transfected cells was used to perform semiquantitative RT-PCR.
The results are expressed as means ± SD. The Mann – Whitney U test was used for all statistical analysis. p < 0.05 was considered significant.
Results and discussion
RA is a heterogeneous autoimmune disease. However, these heterogeneous chronic diseases were recently able to be monitored in line with their gene expression patterns by microarray-based molecular studies . The histology of RA affected joints indicates chronic inflammation with hyperplasia in the synovial lining cells. It is now well established that FLSs actively participate in RA synovitis and that FLSs in RA joints aggressively proliferate to form a pannus, eventually destroying articular bone and cartilage [28, 29]. Several cytokines, such as IL-1β, TNF-α and IL-6, have been described in association with the proliferative response of FLSs. In trials of these therapeutic agents, however, responses were not achieved in a significant proportion of the patients, suggesting that some important factor(s) still remain to be discovered.
To our knowledge this report is the first demonstration that the MLN51 is essential for the hyperproliferation of RA FLSs in line with GM-CSF signaling in RA pathogenesis. Our results show that the SF-mediated growth of RA FLSs was markedly blocked by anti-GM-CSF neutralizing antibody, and additionally that growth-retarded RAFLSs recovered their proliferative capacity by the addition of GM-CSF. These results indicate that GM-CSF in SF is important in the hyperproliferation of RA FLSs. In contrast, in the microarray analysis, semiquantitative RT-PCR and Western blot analysis experiments, we found that the MLN51 was consistently overexpressed in the hyperactive RA FLSs at low passages or the RA FLSs cultured in the presence of SF. MLN51 knock-down by siRNA completely blocked the GM-CSF/SF-mediated proliferation capacity of RA FLSs, suggesting that the MLN51 gene is strongly involved in the pathogenesis of RA.
List of genes upregulated in rheumatoid arthritis/osteoarthritis microarray analysis (HI380)
Genes upregulated in RA fibroblast-like synoviocytesa
RA/OA FLS ratiob
CD1A antigen, α polypeptide
T cell activation
CD3D antigen, δ polypeptide (TiT3 complex)
T cell activation
Highly expressed in immature DCs
T cell activation
B cell activation
CD24 antigen (small cell lung carcinoma cluster 4 antigen)
Unknown for human CD24
T cell activation
CD32 antigen (Fcγ receptor II)
Formation of immune complexes
CD33 antigen (gp67)
Inhibition of myeloid cell proliferation
CD36 antigen (collagen type I receptor, thrombospondin receptor)
Recognition and phagocytosis of apoptotic cells
Adhesion or migration
CD79A antigen (immunoglobulin-associated α)
B cell activation
T cell activation
CD206 antigen (macrophage mannose receptor)
Apoptosis or endocytosis
B cell linker protein (SLP65: BLNK)
B cell activation
Ig superfamily protein (Z39IG)
Formation of immune complexes
T cell transcription factor 4 (TCF-4)
Enhancement of the release of extracellular matrix proteins
IL-1 receptor type I
T cell activation
T and B cell activation
T and mast cell activation
T cell activation
Colony-stimulating factor 2 receptor, β, low-affinity (granulocyte – macrophage); CSF-2RB
Cell cycle regulation
Colony-stimulating factor 3 receptor (granulocyte)
Cell cycle regulation
Methyl-CpG-binding domain protein 1 (MBD1)
Cell cycle regulation
In summary, our results strongly suggest that the MLN51 gene, whose expression depends upon GM-CSF signaling, may have a crucial role in the hyperproliferation of FLSs in the pathogenesis of RA.
We have identified and demonstrated for the first time that the MLN51 is highly expressed in RA FLSs. MLN51 overexpression in the RA FLSs is associated with GM-CSF in the SF of patients with RA. The MLN51 seems to have a critical role in the hyperproliferation of FLSs in RA pathogenesis. MLN51 could be an attractive target for the development of new RA therapeutics.
= bone marrow-derived dendritic cell
= dendritic cell
= Dulbecco's modified Eagle's medium
= fetal calf serum
= fibroblast-like synoviocyte
= granulocyte – macrophage colony-stimulating factor
= monoclonal antibody
- MLN51 :
= metastatic lymph node 51
= rheumatoid arthritis
= synovial fluid
= small interfering RNA
= tumor necrosis factor.
We are grateful to Dr Ho-Youn Kim for helpful comments on this project. This work was supported by an SRC grant (R11-2002-098-01004-0) from the Korea Science and Engineering Foundation through the Rheumatoid Research Center at Catholic University Medical School.
- Hale L, Haynes B: Pathology of rheumatoid arthritis and associated disorders. Arthritis and Allied Conditions: A Textbook of Rheumatology. Edited by: Koopman W. 1997, Baltimore: Williams and Wilkins, 993-1016.Google Scholar
- Okada Y: Proteinases and matrix degradation. Kelly's Textbook of Rheumatology. Edited by: Ruddy S, Harris ED, Sledge JCB. 2001, Philadelphia: WB Saunders, 1: 55-72.Google Scholar
- Mountz JD, Wu J, Cheng J, Zhou T: Autoimmune disease. A problem of defective apoptosis. Arthritis Rheum. 1994, 37: 1415-1420.View ArticlePubMedGoogle Scholar
- Firestein GS, Nguyen K, Aupperle KR, Yeo M, Boyle DL, Zvaifler NJ: Apoptosis in rheumatoid arthritis: p53 overexpression in rheumatoid arthritis synovium. Am J Pathol. 1996, 149: 2143-2151.PubMed CentralPubMedGoogle Scholar
- Nishioka K, Hasunuma T, Kato T, Sumida T, Kobata T: Apoptosis in rheumatoid arthritis: a novel pathway in the regulation of synovial tissue. Arthritis Rheum. 1998, 41: 1-9. 10.1002/1529-0131(199801)41:1<1::AID-ART1>3.0.CO;2-V.View ArticlePubMedGoogle Scholar
- Nakajima T, Aono H, Hasunuma T, Yamamoto K, Shirai T, Hirohata K, Nishioka K: Apoptosis and functional Fas antigen in rheumatoid arthritis synoviocytes. Arthritis Rheum. 1995, 38: 485-491.View ArticlePubMedGoogle Scholar
- Firestein GS, Yeo M, Zvaifler NJ: Apoptosis in rheumatoid arthritis synovium. J Clin Invest. 1995, 96: 1631-1638.PubMed CentralView ArticlePubMedGoogle Scholar
- Baier A, Meineckel I, Gay S, Pap T: Apoptosis in rheumatoid arthritis. Curr Opin Rheumatol. 2003, 15: 274-279. 10.1097/00002281-200305000-00015.View ArticlePubMedGoogle Scholar
- Pap T, Nawrath M, Heinrich J, Bosse M, Baier A, Hummel KM, Petrow P, Kuchen S, Michel BA, Gay RE, et al: Cooperation of Ras- and c-Myc-dependent pathways in regulating the growth and invasiveness of synovial fibroblasts in rheumatoid arthritis. Arthritis Rheum. 2004, 50: 2794-2802. 10.1002/art.20461.View ArticlePubMedGoogle Scholar
- Huber LC, Distler O, Tarner I, Gay RE, Gay S, Pap T: Synovial fibroblasts: key players in rheumatoid arthritis. Rheumatology (Oxford). 2006, 45: 669-675. 10.1093/rheumatology/kel065.View ArticleGoogle Scholar
- Degot S, Regnier CH, Wendling C, Chenard MP, Rio MC, Tomasetto C: Metastatic Lymph Node 51, a novel nucleo-cytoplasmic protein overexpressed in breast cancer. Oncogene. 2002, 21: 4422-4434. 10.1038/sj.onc.1205611.View ArticlePubMedGoogle Scholar
- Degot S, Le Hir H, Alpy F, Kedinger V, Stoll I, Wendling C, Seraphin B, Rio MC, Tomasetto C: Association of the breast cancer protein MLN51 with the exon junction complex via its speckle localizer and RNA binding module. J Biol Chem. 2004, 279: 33702-33715. 10.1074/jbc.M402754200.View ArticlePubMedGoogle Scholar
- Ballut L, Marchadier B, Baguet A, Tomasetto C, Seraphin B, Le Hir H: The exon junction core complex is locked onto RNA by inhibition of eIF4AIII ATPase activity. Nat Struct Mol Biol. 2005, 12: 861-869. 10.1038/nsmb990.View ArticlePubMedGoogle Scholar
- Shibuya T, Tange TO, Stroupe ME, Moore MJ: Mutational analysis of human eIF4AIII identifies regions necessary for exon junction complex formation and nonsense-mediated mRNA decay. RNA. 2006, 12: 360-374. 10.1261/rna.2190706.PubMed CentralView ArticlePubMedGoogle Scholar
- Tange TO, Shibuya T, Jurica MS, Moore MJ: Biochemical analysis of the EJC reveals two new factors and a stable tetrameric protein core. RNA. 2005, 11: 1869-1883. 10.1261/rna.2155905.PubMed CentralView ArticlePubMedGoogle Scholar
- Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, Healey LA, Kaplan SR, Liang MH, Luthra HS, et al: The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 1988, 31: 315-324.View ArticlePubMedGoogle Scholar
- Yoo SA, Bae DG, Ryoo JW, Kim HR, Park GS, Cho CS, Chae CB, Kim WU: Arginine-rich anti-vascular endothelial growth factor (anti-VEGF) hexapeptide inhibits collagen-induced arthritis and VEGF-stimulated productions of TNF-α and IL-6 by human monocytes. J Immunol. 2005, 174: 5846-5855.View ArticlePubMedGoogle Scholar
- Hwang SY, Kim JY, Kim KW, Park MK, Moon Y, Kim WU, Kim HY: IL-17 induces production of IL-6 and IL-8 in rheumatoid arthritis synovial fibroblasts via NF-κ B- and PI3-kinase/Akt-dependent pathways. Arthritis Res Ther. 2004, 6: R120-R128. 10.1186/ar1038.PubMed CentralView ArticlePubMedGoogle Scholar
- Min SY, Hwang SY, Jung YO, Jeong J, Park SH, Cho CS, Kim HY, Kim WU: Increase of cyclooxygenase-2 expression by interleukin 15 in rheumatoid synoviocytes. J Rheumatol. 2004, 31: 875-883.PubMedGoogle Scholar
- Lutz MB, Kukutsch N, Ogilvie AL, Rossner S, Koch F, Romani N, Schuler G: An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J Immunol Methods. 1999, 223: 77-92. 10.1016/S0022-1759(98)00204-X.View ArticlePubMedGoogle Scholar
- Yanagawa Y, Iijima N, Iwabuchi K, Onoe K: Activation of extracellular signal-related kinase by TNF-alpha controls the maturation and function of murine dendritic cells. J Leukoc Biol. 2002, 71: 125-132.PubMedGoogle Scholar
- Winzler C, Rovere P, Rescigno M, Granucci F, Penna G, Adorini L, Zimmermann VS, Davoust J, Ricciardi-Castagnoli P: Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures. J Exp Med. 1997, 185: 317-328. 10.1084/jem.185.2.317.PubMed CentralView ArticlePubMedGoogle Scholar
- Ahn JH, Lee Y, Jeon C, Lee SJ, Lee BH, Choi KD, Bae YS: Identification of the genes differentially expressed in human dendritic cell subsets by cDNA subtraction and microarray analysis. Blood. 2002, 100: 1742-1754.PubMedGoogle Scholar
- Yang YH, Dudoit S, Luu P, Lin DM, Peng V, Ngai J, Speed TP: Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res. 2002, 30: e15-10.1093/nar/30.4.e15.PubMed CentralView ArticlePubMedGoogle Scholar
- Yang D, Buchholz F, Huang Z, Goga A, Chen CY, Brodsky FM, Bishop JM: Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells. Proc Natl Acad Sci USA. 2002, 99: 9942-9947. 10.1073/pnas.152327299.PubMed CentralView ArticlePubMedGoogle Scholar
- Calegari F, Haubensak W, Yang D, Huttner WB, Buchholz F: Tissue-specific RNA interference in postimplantation mouse embryos with endoribonuclease-prepared short interfering RNA. Proc Natl Acad Sci USA. 2002, 99: 14236-14240. 10.1073/pnas.192559699.PubMed CentralView ArticlePubMedGoogle Scholar
- Oertelt S, Selmi C, Invernizzi P, Podda M, Gershwin ME: Genes and goals: an approach to microarray analysis in autoimmunity. Autoimmun Rev. 2005, 4: 414-422. 10.1016/j.autrev.2005.05.004.View ArticlePubMedGoogle Scholar
- Pap T, Muller-Ladner U, Gay RE, Gay S: Fibroblast biology. Role of synovial fibroblasts in the pathogenesis of rheumatoid arthritis. Arthritis Res. 2000, 2: 361-367. 10.1186/ar113.PubMed CentralView ArticlePubMedGoogle Scholar
- Yamanishi Y, Firestein GS: Pathogenesis of rheumatoid arthritis: the role of synoviocytes. Rheum Dis Clin North Am. 2001, 27: 355-371. 10.1016/S0889-857X(05)70206-4.View ArticlePubMedGoogle Scholar
- Campbell IK, Bendele A, Smith DA, Hamilton JA: Granulocyte-macrophage colony stimulating factor exacerbates collagen induced arthritis in mice. Ann Rheum Dis. 1997, 56: 364-368.PubMed CentralView ArticlePubMedGoogle Scholar
- Hamilton JA: GM-CSF in inflammation and autoimmunity. Trends Immunol. 2002, 23: 403-408. 10.1016/S1471-4906(02)02260-3.View ArticlePubMedGoogle Scholar
- Holmdahl R, Jansson L, Andersson M, Larsson E: Immunogenetics of type II collagen autoimmunity and susceptibility to collagen arthritis. Immunology. 1988, 65: 305-310.PubMed CentralPubMedGoogle Scholar
- Banchereau J, Steinman RM: Dendritic cells and the control of immunity. Nature. 1998, 392: 245-252. 10.1038/32588.View ArticlePubMedGoogle Scholar
- Mellman I, Turley SJ, Steinman RM: Antigen processing for amateurs and professionals. Trends Cell Biol. 1998, 8: 231-237. 10.1016/S0962-8924(98)01276-8.View ArticlePubMedGoogle Scholar
- Steinman RM: DC-SIGN: a guide to some mysteries of dendritic cells. Cell. 2000, 100: 491-494. 10.1016/S0092-8674(00)80684-4.View ArticlePubMedGoogle Scholar
- Thomas R, Davis LS, Lipsky PE: Rheumatoid synovium is enriched in mature antigen-presenting dendritic cells. J Immunol. 1994, 152: 2613-2623.PubMedGoogle Scholar
- Summers KL, O'Donnell JL, Williams LA, Hart DN: Expression and function of CD80 and CD86 costimulator molecules on synovial dendritic cells in chronic arthritis. Arthritis Rheum. 1996, 39: 1287-1291.View ArticlePubMedGoogle Scholar
- Pettit AR, Thomas R: Dendritic cells: the driving force behind autoimmunity in rheumatoid arthritis?. Immunol Cell Biol. 1999, 77: 420-427. 10.1046/j.1440-1711.1999.00855.x.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.