Rheumatology 2001; 40: 375-383
© 2001 British Society for Rheumatology
Amelioration of rat antigen-induced arthritis by liposomally conjugated methotrexate is accompanied by down-regulation of cytokine mRNA expression
Department of Rheumatology,
1 Department of Nephrology and
2 Department of Histopathology, University of Wales College of Medicine, Heath Park, Cardiff, UK
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Abstract |
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Objectives. We examined the temporal changes in the expression of interleukin 1ß (IL-1ß), tumour necrosis factor
(TNF-) and interleukin 6 (IL-6) in the rat antigen-induced arthritis (AIA) model and investigated how their expression was modulated following disease amelioration by liposomally conjugated methotrexate (G-MLV).
Methods. On the day of arthritis induction (day 0), rats were treated with a single intra-articular injection of G-MLV, methotrexate (MTX), a dose of lipid equivalent to G-MLV (E-LIPO) or saline. On days 3 and 7 after disease induction, animals from each experimental group were killed. Joint tissue was examined histologically and for mRNA expression (IL-6, IL-1ß and TNF-) using semiquantitative reverse transcription–polymerase chain reaction.
Results. There was no significant difference (ANOVA) in knee swelling between MTX-, E-MLV- or saline-treated animals from day 0 to day 7. By day 1, G-MLV significantly reduced knee swelling (1.94±0.12 mm; P<0.0001) compared with rats treated with MTX (3.17±0.18 mm). G-MLV treatment also significantly inhibited the histological progression of AIA. This reduction in disease severity was accompanied by a reduction in IL-1ß mRNA expression in synovial tissue extracts on day 3 and IL-6 mRNA expression on both day 3 and day 7.
Conclusions. Liposomally conjugated MTX may exert its beneficial effects in experimental arthritis through IL-1ß and IL-6 inhibition.
KEY WORDS: Antigen-induced arthritis, Methotrexate, Rat, Liposomes, Gene expression.
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Introduction |
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According to current knowledge, the development of synovitis in rheumatoid arthritis (RA) is a cell-mediated process involving both infiltrating and resident synovial cell populations [1–3]. Activated macrophages (m
) recruited to the rheumatoid joint are known to secrete various mediators (proteolytic enzymes, eicosanoids, superoxide anion and cytokines) which are thought to participate in various phases of disease induction and tissue injury. Infiltrating m are therefore a possible cellular target for the development of potential therapies of treat RA. Most forms of arthritis, including RA, are incurable and often respond poorly to treatment. This situation lends itself to the development of new anti-arthritis therapies. To this end we suggest the use of liposomes, which are naturally targeted to cells of the mononuclear phagocyte lineage and serve as inert vehicles for the delivery of agents, such as methotrexate (MTX), to the joint.
Intra-articular MTX or glucocorticoid injections for RA therapy have previously been administered with the aim of optimizing the local anti-inflammatory effect at the injection site whilst achieving minimal systemic absorption [4–6]. The efficacy of the intra-articular therapy was, however, reduced by the rapid clearance of the drugs from the joint cavity. This can be overcome by the administration of such agents encapsulated in liposomes [7, 8]. Liposomal delivery of drugs prolongs the residence time of the drug in the joint cavity and has been shown to be successful in man, the rabbit and the rat. Our own experiments using liposomally conjugated MTX (G-MLV), a liposomal formulation of MTX optimized for maximal drug loading and joint retention accompanied by minimal drug leakage after intra-articular administration, produced a rapid and sustained anti-inflammatory effect when used to treat established rat antigen-induced arthritis (AIA) [9]. It was also more potent than an equivalent dose of free MTX in this respect. An in vivo mechanism of action for G-MLV was not identified in this study, although the results of our extensive in vitro experiments suggested that G-MLV's anti-inflammatory effects were mediated through the inhibition of m-derived products [interleukin-1ß (IL-1ß), tumour necrosis factor (TNF-) and prostaglandin E2 (PGE2)] [10, 11] .
The AIA model has been used extensively to elucidate pathogenic mechanisms relevant to RA in humans and to identify potential targets for therapeutic intervention. AIA is induced by a single intra-articular injection of an antigen to animals preimmunized to the same antigen suspended in complete Freunds' adjuvant [12, 13]. It has many pathological features in common with RA in humans, including synovial histopathology, deposition of immune complexes in joint cartilage and pannus formation [13–15]. Furthermore, numerous studies have identified the important role of activated m and their sectretory products [IL-1, TNF- and interleukin 6 (IL-6)] in the pathogenesis of AIA [16, 17]. Consequently, rat AIA was the experimental model of choice for our present study.
In this paper we address the role of the pro-inflammatory cytokines IL-1, TNF- and IL-6 in the pathogenesis of rat AIA by looking at specific cytokine mRNA expression in normal and arthritic joint tissue 3 and 7 days after the induction of arthritis. We also describe, for the first time, the effect of G-MLV treatment upon IL-1ß TNF- and IL-6 mRNA expression in synovial joints.
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Materials and methods |
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Liposome preparation
Control liposomes (E-MLV) were produced by following the method originally described by Bangham et al. in 1965 [18]. Briefly, dry phospholipid films were prepared using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol and 1,2-dimyristoyl-sn-glycero-3-phosphate, sodium salt to a molar ratio of 7:2:1 (Northern Lipids, Vancouver, Canada). Multilamellar vesicles were produced upon addition of 0.9% (w/v) saline.
Liposomal preparations containing MTX-
-dimyristoylphosphatidylethanolamine (DMPE) were formed by mixing predetermined quantities of conjugate and 7:2:1 lipid in chloroform and methanol (1:1). Large multilamellar liposomes (G-MLV; 1.2 µm) containing MTX--DMPE were prepared as described above.
Animals
Male inbred Lewis rats (180–200 g) were obtained from Bantin and Kingman (The Field Station, Grimston, Hull, UK). The animals were housed in cages of five, were allowed food and water ad libitum and were kept in the Biomedical Services Department for 1 week prior to their first immunization. The animals were housed in a cycle of 12 h light/12 h darkness.
Arthritis induction
Arthritis was induced in the right knee joint of each rat by a method described previously [9]. Briefly, on two occasions a week apart, male Lewis rats (200 g) were injected s.c. with an emulsion of equal volumes of methylated bovine serum albumin (mBSA; 0.5 mg; Sigma Chemical, St Louis, MO, USA) and Freund's complete adjuvant (0.25 mg Mycobacterium tuberculosis; Sigma). Fourteen days after the second immunization, the rats were given an intra-articular injection of mBSA (50 µg) into the right knee joint. One day after this inducing dose the rats developed inflammation in the right knee joint. The progression of inflammation was monitored at regular intervals by measuring knee diameters (mean of four readings), with the joint flexed at an angle of 90°, using a digital micrometer.
Treatment of AIA
On the day of arthritis induction (day 0), rats were injected with 100 µl of test agent (spiked with 50 µg mBSA) into the right knee (inflamed) and 100 µl saline (0.9% w/v) into the left knee (normal). One group of rats was treated with G-MLV (n=18) at a dose of MTX and lipid equivalent to 500 µg and 10 µmol respectively. The remaining rats received either free MTX (n=9; 500 µg; Sigma), E-MLV (n=18; 10 µmol total lipid) or saline (n=18; 0.9% w/v). Joint swelling (right knee diameter minus left knee diameter) measurements were made to assess the effect of the respective treatments upon joint inflammation. Animals from the individual treatment groups were killed on either day 3 or day 7 and their joint tissue was reserved for histological processing or determination of the time-dependent changes in cytokine mRNA expression.
Histological grading of inflammation
Rats from different treatment groups were killed either 3 or 7 days after arthritis induction. The knee joints were removed in toto, fixed in formalin-buffered saline, decalcified, routinely processed and embedded in paraffin wax 2 weeks before histological processing. Sections (4 µm thick) were cut in the sagittal plane and stained with haematoxylin/eosin. At least three section levels per joint were examined to ensure all the measurements and scoring were performed on approximately the same level within the joint. All sections were coded prior to assessment to eliminate observer bias and subsequently scored by two independent observers. The histological parameters assessed were synovial lining thickness, inflammatory infiltrate, intraluminal inflammatory exudate, synovial vascularity and number of cartilaginous/bony erosions. The synovial thickness was measured by an eye-piece scale on a total of eight high-power microscopic fields, four on each side of the joint cavity. The sum of the scored parameters, shown in Table 1
, formed the overall arthritis index (AI) for each section.
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Statistics
One-way analysis of variance (ANOVA) was used to determine whether knee swelling was significantly different between MTX-, G-MLV-, E-MLV- and saline-treated rats from day 0 (baseline) to day 7. The modified Student's t-test (Bonferroni method for multiple comparisons) was used to identify which groups were different.
Molecular biology
The time-dependent changes in mRNA expression for IL-6, IL-1ß and TNF- were determined on day 3 and day 7 using reverse transcription–polymerase chain reaction (RT-PCR). Total cellular RNA was isolated by lysing synovial tissue isolated from normal or arthritic knee joints with 1.0 ml of RNAzol B (Biogenesis, Poole, UK). One hundred microlitres of chloroform:isoamyl alcohol (24:1) was added and the mixture shaken for 30 s. Extracts were incubated for 5 min at 4°C to allow the phases to separate. After centrifugation at 11 000 g the aqueous phase was mixed with an equal volume of isopropanol and RNA was precipitated at -70°C for 24 h. Total synovial RNA (1 µg) was denatured at 95°C for 5 min in the presence of 100 pmol random hexamers pd[N]6 (Pharmacia Biosystems, Milton Keynes, UK), and cooled on ice for 2 min. The RNA was reverse-transcribed in a 20 µl final volume of 1xPCR buffer (10 mM Tris, pH 8.3, 50 mM KCI, 1.5 mM MgCl2, 0.001% gelatin), 6.25 µM of each deoxynucleoside triphosphate (dNTP), 20 U of RNAsin (Gibco/BRL Life Technologies, Paisley, UK), 10 mM dithiothreitol and 200 U of reverse transcriptase (Gibco/BRL Life Technologies). The reaction mixture was incubated at room temperature for 10 min, then at 42°C for 40 min and at 95°C for 5 min.
The cDNA (2 µl) product was PCR-amplified in 50 µl final volume of 10 mM Tris, pH 8.3, 50 mM KCI 1.5 mM MgCl2, 200 µM dNTP, 0.001% gelatin, 0.5 µM each primer and 1.25 U of AmpliTaq DNA polymerase (Applied Biosystems, Warrington, UK) in a Perkin-Elmer 480 thermocycler polymerase (Applied Biosystems). Heating to 72°C for 10 min was followed by one cycle of 94°C for 3 min, 55°C for 1 min and 72°C for 1 min. PCR optimization protocols were performed for each cytokine primer set. Amplification was within the exponential phase of the reaction at 35 cycles for -actin and IL-6, 34 cycles for IL-1ß 37 cycles for TNF-. Each cycle comprised heating to 94°C for 40 s, 55°C for 1 min and 72°C for 1 min. Each sample was finally subjected to 1 cycle of 94°C for 40 s and 60°C for 10 min.
Primers for -actin, IL-6, IL-1ß and TNF- were designed from the published cDNA sequences and were obtained from Genosys (Cambridge, UK): -actin: 3'-GGAGCAATGATCTTGATCTT-5', 5'-TCCTGAGGTACGGGTCCTTCC-3', product size 204 base pairs (b.p.); IL-6: 5'-CTTGGGACTGATGTTGTTGAC-3', 5'-GAAGTTGGGGTAGGAAGGAC-3', product size 468 b.p.; IL-1ß: 5'-TCAAGGCATAACAGGCTCATC-3', 5'-CCACGGGCAAGACATAGGTAG-3', product size 315 b.p.; TNF-: 5'-CGGGGGCCACCACGCTCTTC-3', 5'-GGCAAATCGGCTGACGGTGTG-3', product size 362 b.p.
PCR product (10 µl) was electrophoresed on a 3% agarose gel, stained with ethidium bromide and photographed. Negatives were scanned using a densitometer (BioRad 670; BioRad Laboratories, Hemel Hempstead, UK) and the densities of the bands compared with those of the housekeeping gene (-actin). Results were expressed as ratios compared with -actin.
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Treatment of AIA
On day 0 (day of arthritis induction) one group of rats were treated with a single intra-articular dose of G-MLV (equivalent to 500 µg MTX), and inflammation was monitored by performing knee-swelling measurements over a 7-day experimental period (Fig. 1
). Separate control groups of animals were given free MTX (500 µg), E-LIPO (equivalent lipid dose to G-MLV) or saline. When these three control groups were compared there was no significant difference (ANOVA) in knee swelling from day 1 (3.17±0.18, 2.92±0.17 and 3.34±0.16 mm respectively) to day 7 (1.47±0.1, 1.21±0.22 and 1.13±0.15 mm respectively). Therefore knee swelling data for G-MLV treated rats were compared with those for MTX-treated rats to determine whether intra-articular injection of liposomally conjugated MTX--DMPE could modify the course of the mono-articular arthritis.
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One day after treatment, knee swelling (mean±S.E.M.) in G-MLV-treated rats (1.94±0.12 mm; P<0.0001) was significantly less than in MTX-treated rats. This difference remained significant at all time points to day 7, when knee swelling was reduced to values comparable with those noted in non-arthritic knees (0.19±0.06 mm; P<0.0001).
Histology
The effects of each treatment protocol upon the histological progression of the arthritis in individual animals on day 3 and day 7 are reported in terms of synovial lining thickness (Fig. 2) and arthritis index (Fig. 3). There was no significant difference in synovial lining thickness (µm, mean±S.E.M.) between E-MLV-treated animals, MTX-treated animals and saline-treated arthritic controls either on day 3 [29.23±6.54 (n=5), 41.9±8.6 (n=3) and 21.14±1.49 (n=5) respectively] or on day 7 [46.92±15.94 (n=5), 22.92±3.92 (n=5) and 24.72±1.91 (n=5) respectively]. In contrast, G-MLV treatment resulted in significantly lower values compared with E-MLV (P<0.04, P<0.03), MTX (P<0.007, P<0.01) and saline-treated arthritic controls (P<0.03, P<0.0002) on day 3 [15.8±1.48 (n=5)] and on day 7 (10.46±1.47 (n=5)]. Synovial lining thickness in G-MLV-treated rats was, however, significantly greater than in normal joints on day 3 [5.58±0.33 (n=6), P<0.0001] and day 7 [5.3±0.36 (n=6) P<0.01].
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On day 3 there was no significant difference in overall arthritis index between G-MLV-, E-MLV-, MTX- and saline-treated (arthritic control) animals (ANOVA). However, on day 7 the arthritis index in joints treated with G-MLV [0.41±0.2 (n=6)] was significantly lower than in joints treated with E-MLV [2.5±0.54 (n=6), P<0.003] or MTX [1.4±0.43 (n=5), P<0.05]. In addition, none of the sections from animals treated with G-MLV showed signs of either cartilage destruction or bone erosions compared with six, four and four sections, respectively, from E-MLV-, MTX- and saline-treated (arthritic control) animals. Photographs detailing the major histological changes exerted by G-MLV treatment are shown in Fig. 4
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Cytokine mRNA expression
In separate experiments, rats were killed 3 and 7 days after arthritis induction. Due to Home Office regulations, under the Animal (Scientific Procedures) Act 1986, the minimum number of animals per experimental group (n=3) to attain statistical significance had to be used for these studies. Synovial tissue was carefully separated from cartilage and bone, and IL-6, IL-1ß and TNF- mRNA expression was examined in both normal and arthritic (E-MLV-, G-MLV- and saline-treated) tissue samples.
On day 3, expression of both IL-6 (Fig. 5A) and IL-1ß (Fig. 5) mRNA (expressed as the cytokine/-actin ratios) was significantly reduced in joints treated with G-MLV (IL-6 0.24±0.05, P<0.001; IL-1ß 0.49±0.05, P<0.003) compared with saline-treated arthritic controls (1.79±0.18; 1.06±0.07). Furthermore, in joints treated with G-MLV, mRNA ratios were not significantly different from normal (non-arthritic) values (G-MLV 0.35±0.06; normal 0.43±0.03). TNF- mRNA levels were not significantly different when experimental groups were compared (Fig. 5C).
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On day 7, neither IL-1ß nor TNF-
mRNA levels were significantly different when experimental groups were compared (Fig. 6A and B). The IL-6/-actin mRNA ratio in G-MLV-treated rats (0.98±0.18) was significantly lower than in either E-MLV-treated rats (2.83±0.23, P<0.003) or saline-treated arthritic controls (1.91±0.2, P<0.02) but was not significantly different from normal levels (0.57±0.15) (Fig. 6C).
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Discussion |
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In this paper we report that a single intra-articular injection of liposomally conjugated MTX (G-MLV) ameliorated rat AIA and that this anti-inflammatory effect was accompanied by the inhibition of both IL-6 and IL-1ß mRNA expression in synovial tissue.
G-MLV treatment also resulted in the inhibition of AIA progression. Histological comparisons were made between knee joint sections taken from rats treated with G-MLV, MTX or E-MLV and arthritic controls, on day 3 and day 7 after arthritis induction. Rats treated with either MTX or E-MLV and arthritic controls developed severe arthritis characterized by marked lining layer hyperplasia, dense mononuclear cell infiltration (day 3 only), neovascularization, pannus formation and erosions of articular cartilage and bone. In contrast, only mild arthritis characterized by two or three layers of flat synovial lining cells was observed in sections taken from G-MLV-treated rats. Furthermore, none of the sections isolated from G-MLV-treated rats demonstrated histological changes associated with the erosion of articular cartilage or bone.
The role of IL-1ß in the induction of acute synovitis and the development of chronic arthritis in AIA has been studied extensively. Van de Loo et al. [16] studied the influence of IL-1, TNF- and IL-6 upon cartilage proteoglycan metabolism and destruction and concluded that proteoglycan synthesis in both zymosan-induced and antigen-induced murine arthritis models was due to the combined local action of IL-1 (+ß) and that neither TNF- nor IL-6 was involved. In a more recent paper by Ohshima et al. [19], the expression of IL-1ß, TNF- and IL-6 was demonstrated in the arthritic joints of wild-type (IL-6 +/+) mice. Our results are in agreement with these findings. Furthermore, comparable mRNA expression of both IL-1ß and TNF-, but not IL-6, was detected in the inflamed joints of their IL-6-deficient (IL-6 -/-) littermates. These animals developed a mild arthritis where the articular cartilage was well preserved 35 days after disease induction; in contrast, IL-6 (+/+) mice developed severe arthritis in which the articular cartilage was completely destroyed, indicating that IL-6 may play a role in the erosive phase of murine AIA.
In a study to investigate the influence of cyclosporin A on cytokine levels in synovial fluid and serum of rats with AIA, Bauer et al. [20] demonstrated that IL-6 in the synovial fluid and serum showed good correlation with the severity of the disease. In addition, inhibition of IL-6 by cyclosporin A effectively inhibited the chronic phase of arthritis, as demonstrated by decreased joint swelling and a reduced histological arthritis score.
Taken together, our molecular biology and histology results suggest that both IL-1ß and IL-6 play a role in the development of rat AIA, although this would have to be proven conclusively by using neutralizing anti-cytokine antibodies.
G-MLV treatment did not affect TNF- expression in the joint on either day 3 or day 7. It would, therefore, appear that TNF- is not directly involved in the pathogenic mechanisms of AIA. Our results are in agreement with observations made by other research groups [16, 20].
There is an increasing body of evidence that m and m-derived ‘phagocytic synovial lining cells’ (type A cells) play a key role in initiating and producing cartilage damage in RA and AIA. They have been shown to be a major source of the cytokines (IL-1ß, TNF- and IL-6) responsible for inflammation, inflammatory cell mobilization and tissue destruction [21]. When specifically depleted, in experimental arthritis, the intensity of cytokine production, tissue/cartilage destruction and disease progression is reduced [22, 23]. In RA, synovial fluid levels of IL-6 correlate with synovial lining cellularity [24]. Our present study confirms this relationship between synovial lining cellularity (thickness), inflammation (knee swelling) and cytokine production (mRNA expression) in rat AIA. We have previously reported that G-MLV inhibits the production of m-derived TNF-, IL-1ß and PGE2 in vitro. Thus we may conclude that G-MLV, liposome-mediated therapy, is targeted specifically to m and phagocytic synovial lining cells in vivo, thereby exerting beneficial effects upon the progression of AIA in the rat.
Although the precise mechanisms leading to the development of RA are not entirely understood, it is agreed that cytokines such as IL-1ß, TNF- and IL-6 play an important role. Together these cytokines contribute to the acute and chronic inflammation, cell proliferation and tissue destruction/fibrosis that are characteristic of RA pathology. Recent progress in the field of cytokine research and clinical trials involving anti-cytokine therapy for RA has highlighted the fact that these pro-inflammatory cytokines might be optimal therapeutic targets for RA.
In conclusion, G-MLV administered by intra-articular injection provides a more potent delivery system for MTX in rat AIA. Our present study suggests that G-MLV exerts its beneficial effect by inhibiting the local expression of IL-6 and IL-1ß mRNA.
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Acknowledgments |
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A. S. W. would like to thank the Wales Office of Research and Development for financial support during the course of this study.
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Notes |
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Correspondence to: A. S. Williams, Rheumatology Research Laboratory, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK.
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References |
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