Department of Orthopaedic Surgery,
1 Department of Geriatric Medicine,
2 Department of Gene Therapy Science, Osaka University Medical School,2-2 Yamada-oka, Suita 565-0871, Japan
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Abstract |
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Method. NFB decoy oligonucleotides (ODN) were introduced with the aid of the haemagglutinating virus of Japan (HVJ)liposome method into synovial tissue or synovial cells derived from patients with RA. The levels of interleukin-1ß (IL-1ß), IL-6, TNF-
, intercellular adhesion molecule-1 (ICAM-1) and MMP-1 were determined by means of enzyme-linked immunosorbent assay (ELISA) and Northern blotting analysis. A cell counting kit was used to study the effect of NF
B decoy ODN on synovial cell proliferation.
Results. The production of these mediators was significantly inhibited by the introduction of NFB decoy ODN compared with the effect of scrambled decoy ODN. Transfection of NF
B decoy ODN resulted in a significant inhibition of synovial cell proliferation as compared with that of scrambled decoy ODN.
Conclusion. The results demonstrated in this study suggest the potential usefulness of NFB decoy ODN for gene therapy of inflammatory synovitis of RA.
KEY WORDS: Transcription factor decoy, Gene therapy, Rheumatoid arthritis, HVJ-liposome, Synovial cells.
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Introduction |
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NFB is a heterodimetric DNA-binding protein, and a critical element in the regulation of many genes involved in the complex formed by NF
B and I
B. It has been reported that dissociation of the transcription factor NF
B from this complex plays a pivotal role in the regulation of inflammatory cytokine production by inducing a co-ordinated transactivation of genes including TNF-
, IL-1, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), and intercellular adhesion molecule-1 (ICAM-1) [1418]. In RA, activation of NF
B in synovium has been reported [1921]. Recently NF
B has been reported to regulate matrix metalloproteinase-1 (MMP-1) transcription [22]. In this study we examined the role of NF
B in synovial tissue derived from RA. We hypothesized that the transfection of a sufficient quantity of decoy oligodeoxynucleotides (ODN) containing the NF
B cis element into inflammatory cells in the synovial tissues of RA would effectively bind NF
B, thus preventing those cells from transactivating the gene expression of inflammatory cytokines, adhesion molecules and MMP-1.
To deliver the transcription factor decoy into synovial cells, we used the haemagglutinating virus of Japan (HVJ)liposome-mediated method of gene transfer which has been shown to enhance the efficiency of transcription of oligodeoxynucleotides [2326]. In this study we examined the inhibition of the production of pro-inflammatory cytokines, adhesion molecule and MMP from synovial tissue of patients with RA by the introduction of synthetic double-stranded DNA with high affinity for the NFB binding site.
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Materials and methods |
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Organ culture
The tissue specimen was cut into small pieces, washed three times in phosphate-buffered saline (PBS), and its wet weight determined. Synovial tissues were cultured on 24-well plates at 150 mg/well in Dulbecco's modified Eagle's medium (DMEM) (Gibco BRL, Grand Island, New York, USA) supplemented with 10% fetal calf serum (FCS) (Hyclone Laboratories, Logan, UT, USA).
Synthesis of ODN and selection of sequence targets
The sequences of phosphorothioate double-stranded ODN against the NFB binding site and of scrambled ODN used in this study were reported previously [26]. The phosphorothioate ODN utilized in this study had the following sequences:
NFB ODN (consensus sequences are shown in bold)
5'-CCTTGAAGGGATTTCCCTCC-3'
3'-GGAACTTCCCTAAAGGGAGG-5'
Scrambled ODN
5'-TTGCCGTACCTGACTTAGCC-3'
3'-AACGGCATGGACTGAATCGG-5'
The NFB ODN have been shown to bind the NF
B transcription factor [26]. Synthetic ODN were washed in 70% ethanol, dried and dissolved in sterile Tris-ethylene diamine tetra acetic acid (EDTA) buffer (10 mM Tris, 1 mM EDTA). The supernatant was purified over a nucleic acid purification-10 (NAP-10) column (Pharmacia LKB Biotechnology, Piscataway, NJ, USA), and the ODN concentration was quantitated by spectrophotometry. The single-stranded ODN were annealed for 2 h while the temperature descended from 80 to 25°C.
Gel mobility shift assay
The nuclear extract was prepared from cultured synovial tissues using methods described previously [25]. In brief, synovial tissues were homogenized with Potte-Elvehjem homogenizer in 4 volumes of ice-cold homogenization buffer [10 mM HEPES pH 7.5, 0.5 M sucrose, 0.5 mM spermidine, 0.15 mM spermin, 5 mM EDTA, 0.25 M ethylene glycol tetra acetic acid (EGTA), 7 mM ß-mercaptoethanol, 1 mM phenylmethylsulphonyl fluoride]. After centrifugation at 12 000g for 30 min at 4°C, the pellets were lysed and homogenized in a Dounce homogenizer in 1 volume of ice-cold homogenization buffer containing 0.1% NP-40. They were then centrifuged at 12 000 g for 30 min at 4°C and the pelleted nuclei were washed twice with ice-cold buffer containing 0.35 M sucrose. The nuclei were pre-extracted with 1 volume of ice-cold homogenization buffer containing 0.05 M NaCl and 10% glycerol for 15 min at 4°C. The nuclei were then extracted with homogenization buffer containing 0.3 M NaCl and 10% glycerol for 1 h at 4°C and the concentration of DNA was adjusted to 1 mg/ml. After the nuclear extract was pelleted at 12 000 g for 30 min at 4°C, the supernatant was brought to 45% (NH4)2SO4 and stirred for 30 min at 4°C. The precipitated protein was collected at 17 000 g for 30 min, resuspended in homogenization buffer containing 0.35 M sucrose, and stored in aliquots at -70°C. NFB ODN were labelled as probes at the 3' end by means of a 3' end-labelling kit (Clontech Inc., Palo Alto, CA, USA). After end-labelling, 32P-labelled ODN were purified over a nick column (Pharmacia LKB Biotechnology). Binding reactions (10 µl) including the 32P-labelled probe (0.51 ng, 10 00015 000 c.p.m.), and 1 µg of polydeoxyinosinic-deoxycytidic acid (Sigma Chemical Co., St Louis, MO, USA) were incubated with nuclear extract for 30 min at room temperature and then loaded on to a 5% polyacrylamide gel. The gels were subjected to electrophoresis, dried, and pre-incubated with parallel samples 10 min before the addition of the labelled probe.
Preparation of HVJ-liposomes containing ODN and transfection
Phosphatidylserine, phosphatidylcholine, and cholesterol were mixed in a weight ratio of 1:4.8:2 [2326]. The lipid mixture (10 mg) was deposited on the sides of a flask by removal of tetrahydrofuran in a rotary evaporator. Dried lipid was hydrated in 200 µl of balanced salt solution (BSS; 137 mM NaCl, 5.4 mM KCl, 10 mM Tris-HCl, pH 7.6) containing synthetic double-stranded ODN. Liposomes were prepared by shaking and sonication. Purified HVJ (Z strain) was inactivated by ultraviolet irradiation (110 erg/mm2/s) for 3 min just before use. The liposome suspension (0.5 ml, containing 10 mg of lipids) was mixed with HVJ (10 000 haemagglutinating units) in a total volume of 4 ml of BSS. The mixture was incubated at 4°C for 10 min and then for 60 min with gentle shaking at 37°C. Free HVJ was removed from the HVJ-liposomes by sucrose density gradient centrifugation. The top layer of gradient containing purified HVJ-liposomes was collected for use.
Synovial tissues were placed in a serum-free medium 6 h prior to the transfection, then washed three times with BSS containing 2 mM CaCl2. We transfected decoy ODN into synovial tissues by utilizing the HVJ-liposome method. The HVJ-liposome complex (15 µM of encapsulated ODN) was added to the synovial tissues and the cells were incubated at 37°C for 30 min. Finally, fresh medium was added to the cells, which were incubated in a CO2 incubator.
Estimation of the transfection efficiency
To check the localization of transfected FITC-labelled ODN, cryostat sections of synovium transfected with decoy ODN were prepared for fluorescence microscopy. To identify the localization of FITC-labelled ODN, the sections were also stained with propidium iodide (Sigma Chemical Co.), and observed under an ultraviolet laser scanning confocal microscope (PCM 2000: Nikon, Tokyo, Japan).
RNA extraction and Northern blot analysis
Twenty-four hours after transfection of the HVJ-liposome solution, RNA was extracted from synovial tissues by means of RNAzol (Tel-Test Inc., Friendswood, TX, USA) for Northern blot analysis, which was performed as described previously [25]. Briefly, samples (30 µg) were electrophoresed on a 1.5% agarose formaldehyde denaturing gel and transferred to a nitrocellulose membrane (Amersham International, Amersham, UK). The filter was baked, pre-hybridized and hybridized with 32P-labelled IL-1ß, TNF-, ICAM-1 and GAPDH ODN probes (Clontech Laboratories Inc.). The filters were washed rigorously and then exposed to X-ray film.
Densitometric analysis
The relative intensities of bands of interest were analysed with the use of a NSF-300G scanner (Microtek, Anaheim, CA, USA) and scan analysis software (Biosoft, Palo Alto, CA, USA). The results were expressed as the mean ratio of relative intensities to that of the control (synovial tissues with RA transfected with HVJ-liposome solution alone) (determined by the gel mobility shift assay).
Measurement of generated cytokines, MMP-1 and adhesion molecule by enzyme-linked immunosorbent assay (ELISA)
Three days after transfection, the conditioned medium was collected, centrifuged at 600g for 10 min and stored at -20°C. The synovial tissues were homogenized in 1 ml of PBS buffer and centrifuged and the supernatant was stored at -20°C. Two hundred microlitres of supernatant was used for measurement. The assays for human IL-1ß, IL-6, TNF-, and MMP-1 in the supernatant and human ICAM-1 in synovial tissues were performed by ELISA kit (IL-1ß, IL-6, TNF-
, ICAM-1: R&D Systems Inc, Minneapolis, MN, USA; MMP-1: Amersham International) by following the manufacturer's protocol.
Synovial cell preparation
The synovial specimens were finely minced into small pieces, soaked in a solution of enzyme cocktails containing 0.1% type IV collagenase, 0.1% hyaluronidase, and 0.01% DNase (all from Sigma Chemical Co.), and incubated for 2 h at 37°C in a shaking water bath. After the removal of debris by filtration, the cells thus obtained were suspended in DMEM, washed twice, resuspended with DMEM with 10% FCS, and seeded in culture dishes. After overnight culture, non-adherent cells were removed, while adherent cells were re-cultured. The synovial cells used in the experiments were the third passage.
Cell proliferation assay
Synovial cells were seeded on to uncoated 24-well tissue culture plates (Corning Inc., Corning, NY, USA) at 4000 cells/well. The cells were then incubated in DMEM with 10% FCS for 48 h. After transfection of decoy ODN, the medium was changed to fresh DMEM with 10% FCS. Four days after transfection, an index of cell proliferation was determined by using sulphonated tetrazolium salt, and a 4-[3-(4-iodophenyl)-2- (4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulphonate (WST-1) cell counting kit, which is similar to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay [28]. This compound produces a highly water-soluble formazan dye, which makes the assay procedure easier to perform.
Statistical analysis
All values are expressed as mean ± standard error of the mean (S.E.M.). Analysis of variance with subsequent Dunnet's test was used to determine significant differences in multiple comparisons. P < 0.05 was considered significant. All experiments were carried out at least three times.
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Results |
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NFB activation in synovium derived from RA
We ascertained that increases in NFB binding activity in synovial cells derived from patients with RA. We could also demonstrate by means of the gel mobility shift assay that the NF
B binding activity was enhanced in synovial cells from patients with RA but not in synovial cells from trauma patients (Fig. 2
). This NF
B binding was eliminated by pre-incubation of nuclear extracts with excess amounts of unlabelled NF
B ODN, but not with excess unlabelled scrambled ODN (Fig. 2
).
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Effect of NFB decoy on mRNA expression of IL-1ß, TNF-
, and ICAM-1
We also examined the effect of NFB decoy ODN on the RNA expression level of several cytokines and adhesion molecule genes whose expressions are known to be up-regulated in RA. Total RNA was extracted from synovial tissues 24 h after transfection of ODN and Northern blot analysis was performed using the extracted RNA. As shown in Fig. 3
, RNA expressions of IL-1ß, TNF-
and ICAM-1 in synovial tissues were inhibited by in vitro transfection of NF
B decoy ODN, but not of scrambled decoy ODN.
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Reduction in the binding of NFB activity after decoy transfection
We then examined changes in NFB binding activity after transfection of decoy ODN. Twelve hours after transfection, NF
B decoy ODN reduced NF
B binding activity in nuclear extracts of synovial cells derived from RA patients (Fig. 4A
). This effect was not seen in the case of transfection with scrambled ODN. This reduction was dose dependent of transfected NF
B decoy ODN. A higher concentration (15 µM of decoy ODN) showed a stronger effect than a lower concentration (5 µM). The results of densitometric analysis showed significant differences (Fig. 4B
).
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Effect of NFB decoy on production of cytokines, MMP-1, and ICAM-1
In addition to RNA expression, the protein levels of cytokines and MMP-1 that were secreted into the medium were also studied 72 h after transfection. The levels of IL-1ß (2.7 ± 2.2 ng/ml), IL-6 (2881.1 ± 774.8 pg/ml), TNF- (under detectable level), and MMP-1 (73.8 ± 8.0 ng/ml) in the culture medium of synovial tissues transfected with NF
B decoy ODN were significantly lower than those of synovial tissues transfected with scrambled decoy ODN (IL-1ß: 212.6 ± 43.7 ng/ml; IL-6: 5324.8 ± 474.5 pg/ml; TNF-
: 710.2 ± 107.1 pg/ml; MMP-1: 168.1 ± 23.1 ng/ml) as assessed by ELISA (P < 0.01 for IL-1ß and TNF-
, and P < 0.05 for IL-6 and MMP-1). The level of ICAM-1 in synovial tissues transfected with NF
B decoy ODN (4.2 ± 3.2 ng/ml) was significantly lower than that of synovial tissues transfected with scrambled decoy ODN (36.1 ± 13.3 ng/ml) as assessed by ELISA (P < 0.01) (Fig. 5
).
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Effect of NFB decoy on synovial cell proliferation
Finally, we assessed the effect of NFB decoy ODN on synovial cell proliferation. The level of synovial cell proliferation was determined by using the WST-1 cell counting kit at 4 days after transfection. The index of cell proliferation determined by absorbance at 450 nm using the WST-1 cell counting kit was 0.286 ± 0.28 with synovial cells transfected with NF
B decoy ODN and 0.356 ± 0.41 with synovial cells transfected with scrambled decoy ODN. Transfection of NF
B decoy ODN resulted in a significant inhibition of synovial cell proliferation as compared with scrambled decoy ODN transfected synovial cells and non-transfected synovial cells (P < 0.01) (Fig. 6
).
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Discussion |
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We previously reported that the HVJ-liposome method is an effective ODN delivery system, which enhances the uptake and intracellular stability of ODN in cultured cells [25]. As the decoy effect is apparently located in the nucleus, bypassing the endocytotic pathway and translocation of decoy ODN from the cytoplasm are extremely important. The fusion with the cell membrane mediated by the envelope proteins of HVJ (HN and F proteins) results in intracellular ODN delivery, which bypasses endocytosis. Consequently, this method enhances and prolongs the effect of ODN in vitro. In this study, we confirmed that the HVJ-liposome method is also effective for ODN transfer into synovial cells. We speculate that the enhanced efficiency of this system may result in fewer non-specific toxic effects observed at high concentrations of ODN. Indeed, this method has been used recently for the transfection of plasmid DNA encoding SV 40 large T antigen and ODN into rat knee joints in vivo [35, 36]. As previously shown, the expression of the transgene was recognized in the synovial cells. HVJ-liposome may thus be an effective method for decoy ODN transfer for the purpose of gene therapy for RA.
The progression of arthritis depends upon the activation of the complicated network consisting of cytokines and adhesion molecules. In this study, the production of IL-1ß and TNF- was suppressed to almost below detectable levels, while the production of IL-6 was suppressed by about 55%. Because IL-6 has another transcriptional factor, activator protein-1 (AP-1) [37, 38], only the blockage of NF
B activation may be insufficient for the production of IL-6. MMP-1 directly and efficiently degrades type I, II, and III collagen, and contributes to the destruction of articular cartilage and bone in RA. The inhibitory effect of NF
B decoy on the production of MMP-1 as shown in this study was about 40% of that achieved with the treatment with the scrambled decoy. This is because in the promoter region of MMP-1, AP-1 also plays an important role in transcriptional induction by extracellular stimulation [3941]. For this reason, combination therapy of NF
B decoy and AP-1 decoy could result in a stronger inhibitory effect. Although an inhibitory effect on synovial cell proliferation in vitro was shown in this study, the biological significance of NF
B decoy on synovial proliferation in vivo still remains unknown. The previous in vivo study using an experimental arthritis model showed marked reduction of the weight of synovial tissue in the joints transfected with NF
B decoy compared with the joints transfected with scrambled decoy or untreated joints [36]. These results suggest that NF
B decoy may provide a biologically significant effect on synovial proliferation in vivo.
Here we reported a novel therapeutic strategy to suppress the inflammatory process in synovial cells by transfecting decoy ODN to block the binding of the critical transcription factor NFB to its promoter sequence of the target genes, thereby inhibiting the co-ordinated transactivation of genes of key cytokines and adhesion molecules necessary for the progressive joint destruction in RA. Further studies are necessary to prove that the blockade of those cytokines and adhesion molecules by NF
B decoy ODN is sufficient to block joint inflammation and destruction in vivo. Furthermore, modification of ODN composition in order to prolong decoy stability will be essential for the enhancement of therapeutic efficacy. Despite these limitations, transcription factor decoy technology provides a new tool for identifying biological processes and for treating pathological conditions in the joints. Even though the data derived from in vitro experiments, the results of this study clearly demonstrate that the transcription factor NF
B is one of the key regulators which promote joint destruction in arthritis.
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Acknowledgments |
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Notes |
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References |
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