Effects of combinations of anti-rheumatic drugs on the production of vascular endothelial growth factor and basic fibroblast growth factor in cultured synoviocytes and patients with rheumatoid arthritis

M. Nagashima, K. Wauke, D. Hirano, S. Ishigami, H. Aono1, M. Takai1, M. Sasano1 and S. Yoshino

Department of Joint Disease and Rheumatism, Nippon Medical School, 1-1-5, Sendagi, Bunkyo-ku, Tokyo 113-8603 and
1 Developmental Research Division, Santen Pharmaceutical Co., Ltd, 3-9-19, Shimoshinjo, Higashiyodogawa, Osaka 533-8651, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective. To examine whether different combinations of disease-modifying anti-rheumatic drugs (DMARDs), including bucillamine (BUC), gold sodium thiomalate (GST), methotrexate (MTX), salazosulphapyridine (SASP) and dexamethasone (DEX; a steroid), act by inhibiting the production of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in cultured synoviocytes, causing a decrease in their serum concentrations in patients with rheumatoid arthritis (RA).

Methods. The VEGF and bFGF concentrations in cultured synoviocytes and peripheral blood from patients with RA were measured by enzyme-linked immunosorbent assay and their serum concentrations were measured at two time points.

Results. BUC and GST inhibited VEGF production even when given alone, and a combination of BUC, GST and MTX with DEX also inhibited VEGF production. None of the DMARDs or DEX inhibited bFGF production when given alone, but a combination of SASP and GST inhibited the production of bFGF in cultured synoviocytes. Serum VEGF concentrations were significantly decreased 6 months after the commencement of medication compared with their concentrations before medication.

Conclusion Our results show that the effects of a combination of DEX with any two of BUC, GST, SASP and MTX on the production of VEGF and bFGF in cultured synoviocytes and on the serum concentrations of VEGF in patients with RA may be based on synergistic or additive effects of the drugs.

KEY WORDS: Rheumatoid arthritis, Vascular endothelial growth factor, Basic fibroblast growth factor, Disease-modifying anti-rheumatic drugs.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Angiogenesis, the growth and proliferation of new blood vessels, is important in a variety of pathophysiological processes in rheumatoid arthritis (RA). A number of angiogenic factors are involved in the neovascularization process in the RA joint. These include acidic fibroblast growth factor (aFGF or FGF-1), basic fibroblast growth factor (bFGF or FGF-2), platelet-derived growth factor (PDGF), platelet-derived endothelial cell growth factor and vascular endothelial growth factor (VEGF). These growth factors stimulate vascular endothelial cells in an autocrine and paracrine manner, stimulating angiogenesis [16].

VEGF is a unique peptide growth factor that specifically stimulates the proliferation of endothelial cells [7] and is thought to play an essential role in angiogenesis in a variety of biological processes, including tissue repair and regeneration [8] and tumour growth [911]. We have demonstrated previously that the VEGF polypeptide and mRNA are distributed in the perivascular space and that both are expressed in subsynovial macrophages and synovial lining cells in the synovial tissues of RA patients, by the use of immunohistochemical staining, in situ hybridization and reverse transcription–polymerase chain reaction analysis [12]. Thus, these observations suggest that the constitutive expression of VEGF production in RA may play an important role in the pathophysiology of RA. In addition, bFGF derived from synoviocytes may play a role in stimulating their proliferation and angiogenesis in an autocrine manner in RA [1315].

On the other hand, several disease-modifying anti-rheumatic drugs (DMARDs) have been used to control the progression of RA. While the majority of these DMARDs act as immunomodulatory drugs in RA [1623], some also act by inhibiting cytokines and endothelial cell proliferation [2429]. However, the mechanism underlying the inhibitory effects of DMARDs on angiogenesis remains obscure. By the use of ELISA and reverse transcription–polymerase chain reaction analysis, we have already shown that bucillamine, a DMARD, and dexamethasone when given alone inhibit VEGF production and VEGF mRNA expression, respectively, in cultured synoviocytes [30].

In the present study, we examined whether various combinations of DMARDs, namely bucillamine (BUC), gold sodium thiomalate (sodium aurothiomalate; GST), methotrexate (MTX), salazosulphapyridine (SASP) and dexamethasone (DEX; a steroid), can inhibit the production of VEGF and bFGF in cultured synoviocytes, and our results in vitro are supported by the results of a study in patients with RA.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients and cell preparation
Synovial tissue specimens were obtained from seven patients with RA (stage III or IV) who fulfilled the diagnostic criteria of the American College of Rheumatology and had a disease duration of 10–15 yr. After we had obtained informed consent from the patients, specimens were obtained during synovectomy of the knee or total knee joint arthroplasty and were processed immediately, as described previously [31, 32]. In brief, the synovial tissue was cut into small pieces, washed three times in phosphate-buffered saline (PBS), and treated with 1 mg/ml collagenase (Sigma, St Louis, Missouri, USA) for 30–60 min at 37°C. The cells were suspended in Ham's F-12 medium (Nikken Bio Medical Laboratories, Kyoto, Japan) containing 10% fetal calf serum (FCS; Life Technologies, MD, USA), 100 U/ml penicillin and 100 mg/ml streptomycin. The cell suspension was then plated onto 90-mm culture dishes and cultured under 5% CO2 in a humidified incubator. When the cultured cells reached confluence, they were treated with trypsin and further passaged in other dishes. The cells used for the present experiments were obtained from passages 2–5. Peripheral blood samples were obtained from 129 RA patients and serum was separated from each of the samples by centrifugation at 3000 r.p.m. for 10 min, to examine the correlation between the concentrations of angiogenic growth factors and inflammatory activity in patients with RA.

DMARDs and DEX
Bucillamine was obtained from Santen Pharmaceutical Co. (Osaka, Japan). Salazosulphapyridine, gold sodium thiomalate, methotrexate, and dexamethasone were obtained from Sigma, Shionogi Co. (Osaka, Japan), Nacalai tesque (Kyoto, Japan) and Biomed Res (Plymouth Meeting, PA, USA), respectively. BUC and MTX were used at 10 µg/ml, and GST and SASP were used at 100 µg/ml, while the dose of DEX ranged from 0.1 to 1 µg/ml.

The following combinations of the DMARDs and DEX were used: BUC and SASP; BUC and GST; BUC and MTX; BUC and DEX; SASP and GST; SASP and MTX; SASP and DEX; GST and MTX; GST and DEX; and MTX and DEX.

Analysis of VEGF concentration in the culture supernatants of synoviocytes
For the assay of VEGF production, 24-well flat-bottomed microtitre plates containing the culture medium were seeded with 5 x 104 cells per well. After 24 h, cell growth was arrested by replacing the culture medium with Ham's F-12 medium containing 1% FCS. In the next step, lipopolysaccharide (LPS; Difco Laboratories, Detroit, MI, USA), with or without the DMARDs, was added, followed by incubation for another 72 h and collection of the supernatant. The concentration of VEGF in the supernatant was measured using a VEGF enzyme-linked immunosorbent assay (ELISA) kit (Immuno-Biological Lab Co., Gunma, Japan), and absorbance at 450 nm was recorded using an ELISA plate reader (Bio-Rad, CA, USA).

Analysis of bFGF concentration in the synovial cell lysates
Cells (5 x 104) were plated onto each well of 24-well flat-bottomed microtitre plates in 1 ml of Ham's F-12 medium containing 1% FCS. The culture supernatant was harvested after 3 days of culture in the presence of 10 µg/ml LPS with or without the DMARDs. After washing with PBS, the synovial cells were resuspended in 0.5 ml of 1 M NaCl and 10 mM Tris–HCl (pH 7.5), then frozen at -80°C and thawed. This procedure was repeated three times. After sonication, the concentration of bFGF was measured in these cells using a bFGF ELISA kit (R&D Systems, Minneapolis, MN, USA), and absorbance was recorded at 450 nm using an ELISA plate reader (Bio-Rad).

Analysis of VEGF and bFGF concentrations in the serum of patients with RA
The VEGF and bFGF concentrations in the serum of 129 patients with RA were measured using VEGF and bFGF ELISA kits, to examine the correlations of the serum concentrations of VEGF and bFGF with the serum concentrations of C-reactive protein (CRP) and the Westergren erythrocyte sedimentation rate (ESR). The serum concentrations of VEGF and bFGF in 25 patients with RA were also measured before and after the commencement of treatment with the various combinations of DMARDs, to investigate the therapeutic efficacy of these combinations.

Statistical analysis
All values were expressed as mean ± S.E.M. Differences between groups were tested for statistical significance using Student's t-test, Dunnett's test and the Scheffé test. A P value of <0.05 or <0.01 was considered to denote a significant difference. Correlation coefficients were measured using Pearson's correlation analysis.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Inhibition of VEGF production in the culture supernatant
We have already reported that LPS as well as interleukin (IL)-1ß activates the production of VEGF in the cultured synoviocytes of patients with RA and OA [30]. For cultured synoviocytes in Ham's F-12 containing 1% FCS, we added 10 µg/ml LPS and further incubated the culture for 72 h. The concentrations of VEGF in the culture supernatants were measured using VEGF ELISA kits. The concentrations of VEGF increased with time from 4 to 72 h. Then, to cultured synoviocytes in Ham's F-12 containing 1% FCS and 10 µg/ml LPS, the different combinations of DMARDs were added, followed by further incubation of the cultures for 72 h.

The mean VEGF concentration in stimulated synoviocytes that were not treated with DMARDs was 411.3 ± 67.5 pg/ml. When used alone, BUC and GST (DMARDs) and DEX significantly reduced the VEGF concentration to 184.9 ± 36.0, 138.4 ± 14.4 and 178.4 ± 25.3 pg/ml respectively. In contrast, SASP and MTX did not inhibit VEGF production when used alone (Fig. 1Go). All combinations containing BUC, GST and DEX significantly inhibited VEGF production. Nevertheless, combinations including SASP with MTX did not significantly inhibit VEGF production (Fig. 1Go).



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FIG. 1. Effect of DMARDs on VEGF production by LPS-stimulated synovial cells from eight patients with RA. Results are mean ± S.E. *, P < 0.05; **, P < 0.01 compared with untreated synoviocytes (control); ##, P < 0.01 compared with synoviocytes treated with a single drug. All combinations except the combination of SASP with MTX significantly inhibited VEGF production.

 
In the next step, we examined whether the inhibitory effect of a combination of BUC with DEX on VEGF production was maintained when the concentrations of the drugs were decreased. When the concentration of BUC was not changed but that of DEX was reduced, the combination was still capable of inhibiting VEGF production (Fig. 2Go).



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FIG. 2. Effect of the combination of BUC with DEX on VEGF production by LPS-stimulated synovial cells from eight patients with RA. *, P < 0.05; **, P < 0.01 compared with synoviocytes not treated with BUC or DEX. Both BUC and DEX inhibited VEGF production even when given alone. The efficacy of the combination of BUC with DEX was dependent on their concentrations.

 

Inhibition of bFGF production in cell lysates
The production of bFGF and VEGF production was stimulated in synovial lysates by 10 µg/ml LPS. However, none of the DMARDs or DEX inhibited bFGF production, whether given alone or in combination, except the combination of SASP with GST. This combination decreased the bFGF concentration in the cell lysates significantly from 2768.8 ± 451.9 to 1062.7 ± 288.3 pg/105 cells. The combination of DEX with GST also slightly decreased the concentration of bFGF in the cell lysate (Fig. 3Go).



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FIG. 3. Effect of DMARDs on bFGF production by LPS-stimulated synovial cells from eight patients with RA. Data are mean ± S.E. *, P < 0.05; **, P < 0.01 compared with untreated synoviocytes (control); #, P < 0.05 compared with synoviocytes treated with a single drug. The combinations of GST with SASP and of GST with DEX inhibited bFGF production.

 

Correlation of serum concentrations of VEGF and bFGF with ESR and serum concentration of CRP
We investigated whether the serum concentrations of VEGF and bFGF were correlated with ESR and the serum concentration of CRP as indicators of inflammatory activity in patients with RA. The VEGF concentration in all 129 cases was correlated with ESR and the serum concentration of CRP (Fig. 4Go), but the bFGF concentrations in the sera were not correlated with these parameters (Fig. 5Go). In order to examine whether the effects in vitro reflected those in vivo, the serum concentrations of VEGF and bFGF of 25 patients were measured before and 6 months after the commencement of medication (a combination of prednisolone 5 mg with BUC 100 mg, SASP 1000 mg or MTX 5–7.5 mg). The VEGF concentrations in the sera decreased significantly from 30.73 ± 17.26 pg/ml before medication to 19.58 ± 14.45 pg/ml 6 months after the commencement of medication. On the other hand, serum bFGF concentration decreased non-significantly from 7.25 ± 15.88 pg/ml before medication to 5.73 ± 9.63 pg/ml 6 months after the commencement of medication (Fig. 6Go).



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FIG. 4. Correlation of serum concentration of VEGF with CRP and ESR in patients with RA. Both correlations were significant.

 


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FIG. 5. Correlation of serum concentration of bFGF with CRP and ESR in patients with RA. Neither correlation was significant.

 


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FIG. 6. Serum concentrations of VEGF and bFGF in patients with RA before and 6 months after the commencement of medication. VEGF concentration decreased significantly from 30.73 ± 17.26 pg/ml before medication to 19.58 ± 14.45 pg/ml 6 months after the commencement of medication (P < 0.01), consistent with the results in vitro. The bFGF concentration was not significantly different before and 6 months after the commencement of medication.

 


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
RA has been recognized as an angiogenic disorder that is similar to diabetic retinopathy, fibrovascular disease and tumour growth [33]. In fact, several angiogenic growth factors and inhibitors have been recognized. RA is an angiogenesis-dominant disorder from the viewpoint of the balance between the production of angiogenic growth factors and inhibitors. DMARDs, besides controlling the progression of RA, retard the progression of joint destruction in the majority of patients with early RA [34]. It has been reported that it is possible to retard the rate of erosion of the articular joint [35, 36].

In our previous study, we recognized that RA is an angiogenesis-dominant disease, and we now consider that it is important to investigate whether DMARDs can inhibit the synthesis of angiogenic growth factors (e.g. VEGF and bFGF). Of the DMARDs, BUC and GST inhibited VEGF production in cultured synoviocytes even when given alone. The mechanism underlying the inhibitory effect of BUC on VEGF may depend on its suppressive effect on the transcription of VEGF mRNA [30]. Therefore, we propose that the anti-rheumatic effect of BUC is mediated by the inhibition of VEGF production and angiogenesis.

We examined the inhibitory effects of several combinations of DMARDs on VEGF and bFGF production in cultured synoviocytes, because these drugs have been used in several combinations to control the clinical activity and progression of inflammatory activity in patients with RA. We tried to identify the most effective combinations of DMARDs from the point of view of their inhibitory effect on the production of angiogenic growth factors such as VEGF and bFGF. Our results revealed that combinations containing BUC, GST and DEX inhibit VEGF production. But MTX and SASP, which have antifolate properties [37], have no significant inhibitory effect on VEGF production, whether used alone or in combination.

MTX has been reported to inhibit the binding of IL-1ß to its receptor [19] and the production of IL-6 [38, 39], because MTX leads to increased adenosine release; adenosine then binds to its receptors on macrophages and monocytes, thereby inhibiting the production of several cytokines, such as TNF-{alpha}, IL-6 and IL-8 [40, 41]. But no study has addressed the question of the molecular mechanism by which MTX treatment modulates cytokine release. Nevertheless, a number of hypotheses have been proposed [42].

In regard to combination therapy, while both BUC and DEX have the same effect (inhibiting VEGF production even when given alone), if the concentration of BUC is not changed and that of DEX is gradually reduced, the inhibitory effect on VEGF production is maintained (Fig. 2Go). This result shows that BUC and DEX exert additive effects when used in combination. Paulus reported that combinations of drugs with different toxicities or the use of lower doses of toxic drugs in combination may decrease the risks associated with DMARD therapy while maintaining or increasing their efficacy [43].

Only the combination of SASP with GST had a significant inhibitory effect on bFGF production; the combination of DEX with GST had a slight inhibitory effect. Neither SASP nor GST inhibited bFGF production when given alone, but when they were given in combination they did inhibit bFGF production. These results show that combination therapy rests on two or three factors: synergistic or additive effects, and a mechanism dependent on the drug combination.

We investigated whether the serum concentrations of VEGF and bFGF were correlated with ESR and the serum concentration of CRP, which are indicators of inflammatory activity in patients with RA. It has been reported that there is a significant correlation between the serum concentrations of CRP and IL-6 [44, 45]. Paleolog et al. [46] reported that the serum concentration of CRP is correlated with the concentrations of both VEGF and IL-6 in the serum. Our results show that the serum concentration of VEGF was correlated with the ESR and serum concentrations of CRP, whereas that of bFGF was not related to either of these parameters (Figs 4Go and 5Go). As shown in Fig. 6Go, the serum concentration of VEGF had decreased significantly by 6 months after the commencement of medication compared with the concentration before medication. On the other hand, the concentration of bFGF before medication was not significantly different from that 6 months after the commencement of medication. The same results were thus observed in vitro and in vivo. In our previous study, we reported that the VEGF and bFGF concentrations in joint fluid were significantly higher than those in peripheral blood [47] and that VEGF was produced mainly by macrophages, fibroblasts and synovial cells in the synovial tissues in RA patients [12]. BUC and DEX decrease the VEGF concentrations in the joint fluid and peripheral blood as a result of their inhibitory effect on VEGF production. The concentrations of DMARDs used in the cultured synoviocytes are also sufficient for use in vivo. So we propose that one of the mechanisms of the anti-rheumatic actions of DMARDs is the inhibition of VEGF production, and the inhibition of pathological angiogenesis in RA.

On the other hand, it is unclear how bFGF acts in the pathogenesis of RA. But because bFGF mRNA has been reported to be expressed in synovial lining cells, macrophages, smooth muscle cells, fibroblasts and endothelial cells [13], bFGF may participate in the proliferation of synovial cells in synovial tissue in an autocrine or paracrine manner or in the immune growth network through T cells [15]. The consequences of inhibition of bFGF production by the combination of SASP and GST are the direct inhibition of cell proliferation and the indirect inhibition through the immune growth network.

In summary, our results show for the first time that, in regard to the control of angiogenesis in cultured synoviocytes, both BUC and GST have an inhibitory effect on VEGF peptides and mRNA even when given alone, and that combinations of any two of the drugs BUC, GST, SASP, MTX and DEX, except the combination of MTX with SASP, also have inhibitory effects on VEGF production. These in vitro results were supported by observations in the peripheral blood of patients with RA.


    Acknowledgments
 
This work was supported by a Grant-in-Aid for Scientific Research, from the Ministry of Education, Science, Sports and Culture of Japan (No. 10670433).


    Notes
 
Correspondence to: M. Nagashima, Department of Joint Disease and Rheumatism, Nippon Medical School, 1-1-5, Sendagi, Bunkyo-ku, Tokyo 113–8603, Japan. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Folkman J, Shing Y. Angiogenesis. J Biol Chem1992;267:10931–4.[Free Full Text]
  2. Leung DW, Cachianes G, Kuang W-J, Goeddel DV, Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science1989;246:1306–12.[ISI][Medline]
  3. Sano H, Forough R, Maier JA et al. Detection of high levels of heparin binding growth factor-1 (acidic fibroblast growth factor) in inflammatory arthritic joints. J Cell Biol1990;110:1417–26.[Abstract]
  4. Qu Z, Huang XN, Ahmadi P et al. Expression of basic fibroblast growth factor in synovial tissue from patients with rheumatoid arthritis and degenerative joint disease. Lab Invest1995;73:339–46.[ISI][Medline]
  5. Takeuchi M, Otsuka T, Matsui N et al. Aberrant production of gliostatin/platelet-derived endothelial cell growth factor in rheumatoid synovium. Arthritis Rheum1994;37:662–72.[ISI][Medline]
  6. Remmers EF, Sano H, Lafyatis R et al. Production of platelet derived growth factor B chain (PDGF-B/c-sis) mRNA and immunoreactive PDGF B-like polypeptide by rheumatoid synovium: coexpression with heparin binding acidic fibroblast growth factor-1. J Rheumatol1991;18: 7–13.[ISI][Medline]
  7. Ferrara N, Henzel WJ. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun1989;161:851–8.[ISI][Medline]
  8. Shweiki D, Itin A, Neufeld G, Gitay-Goren H, Keshet E. Patterns of expression of vascular endothelial growth factor (VEGF) and VEGF receptors in mice suggest a role in hormonally regulated angiogenesis. J Clin Invest1993;91:2235–43.[ISI][Medline]
  9. Morii K, Tanaka R, Washiyama K, Kumanishi T, Kuwano R. Expression of vascular endothelial growth factor in capillary hemangioblastoma. Biochem Biophys Res Commun1993;194:749–55.[ISI][Medline]
  10. Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature1992;359:843–5.[ISI][Medline]
  11. Aiello LP, Avery RL, Arrigg PG et al. Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N Engl J Med1994;331:1480–7.[Abstract/Free Full Text]
  12. Nagashima M, Yoshino S, Ishiwata T, Asano G. Role of vascular endothelial growth factor in angiogenesis of rheumatoid arthritis. J Rheumatol1995;22:1624–30.[ISI][Medline]
  13. Nakashima M, Eguchi K, Aoyagi T et al. Expression of basic fibroblast growth factor in synovial tissue from patients with rheumatoid arthritis: detection by immunohistological staining and in situ hybridization. Ann Rheum Dis1994;53:45–50.[Abstract]
  14. Melnyk VO, Shipley GD, Sternfeld MD, Sherman L, Rosenbaum JT. Synoviocytes synthesize, bind and respond to basic fibroblast growth factor. Arthritis Rheum1990;33:493–500.[ISI][Medline]
  15. Byrd V, Zhao X-M, McKeehan WL, Miller GG, Thomas JW. Expression and functional expansion of fibroblast growth factor receptor T cells in rheumatoid synovium and peripheral blood of patients with rheumatoid arthritis. Arthritis Rheum1996;39:914–22.[Medline]
  16. Hashimoto K, Lipsky PE. Immunosuppression by the disease modifying antirheumatic drug bucillamine: Inhibition of human T lymphocyte function by bucillamine and its metabolites. J Rheumatol1993;20:953–7.[ISI][Medline]
  17. Sanders KM, Carlson PL, Littman BH. Effects of gold sodium thiomalate on interferon stimulation of C2 synthesis and HLA-DR expression by human monocytes. Arthritis Rheum1987;30:1032–9.[ISI][Medline]
  18. Gubner R, August S, Ginsberg V. Therapeutic suppression of tissue reactivity. II. Effect of aminopterin in rheumatoid arthritis and psoriasis. Am J Med Sci1951;221:176–82.[ISI]
  19. Brody M, Bohm I, Bauer R. Mechanism of action of methotrexate: experimental evidence that methotrexate blocks the binding of interleukin-1b to the interleukin-1 receptor on target cells. Eur J Clin Chem Clin Biochem1993;31:667–74.[ISI][Medline]
  20. Seitz M, Dewald B, Ceska M, Gerber NJ, Baggiolini M. Interleukin-8 in inflammatory rheumatic diseases: synovial fluid levels, relation to rheumatoid factors, production by mononuclear cells, and effects of gold sodium thiomalate and methotrexate. Rheumatol Int1992;12:159–64.[ISI][Medline]
  21. Seitz M, Loetscher P, Dewald B et al. Methotrexate action in rheumatoid arthritis: stimulation of cytokine inhibitor and inhibition of chemokine production by peripheral blood mononuclear cells. Br J Rheumatol1993;34:602–9.
  22. Carlin G, Djursater R, Smedegard G. Sulfasalazine inhibition of human granulocyte activation by inhibition of second messenger compounds. Ann Rheum Dis1992;51:1230–6.[Abstract]
  23. Carlin G, Djursater R, Smedegard G. Inhibitory effects of sulfasalazine and related compounds on superoxide production by human polymorphonuclear lymphocytes. Pharmacol Toxicol1989;65:121–7.[ISI][Medline]
  24. Koch AE, Cho M, Burrows J, Leibovich SJ, Polverini PJ. Inhibition of production of macrophage-derived angiogenic activity by the anti-rheumatic agents gold sodium thiomalate and auranofin. Biochem Biophys Res Commun1988;154:205–12.[ISI][Medline]
  25. Kouda M, Yoshino S, Nakamura H, Asano G. Effects of disease modifying antirheumatic drugs (DMARDs) and DEX on IL-1ß and IL-6 production by IL-1ß stimulated synovial culture cells. J Nippon Med Sch1996;63:1–5.
  26. Matsubara T, Ziff M. Inhibition of human endothelial cell proliferation by gold compounds. J Clin Invest1987;79:1440–6.[ISI][Medline]
  27. Hirata S, Matsubara T, Saura R, Tateishi H, Hirohata K. Inhibition of in vitro vascular endothelial cell proliferation and in vivo neovascularization by low dose methotrexate. Arthritis Rheum1989;32:1065–73.[ISI][Medline]
  28. Grootveld M, Blake DR, Sahinoglu T. Control of oxidative damage in rheumatoid arthritis by gold(I)-thiolate drugs. Free Radical Res Commun1990;10:199–200.[ISI][Medline]
  29. Matsubara T, Saura R, Hirohata K, Ziff M. Inhibition of human endothelial cell proliferation in vitro and neovascularization in vivo by D-penicillamine. J Clin Invest1989;83:158–67.[ISI][Medline]
  30. Nagashima M, Yoshino S, Aono H, Takai M, Sasano M. Inhibitory effects of anti-rheumatic drugs on vascular endothelial growth factor in cultured rheumatoid synovial cells. Clin Exp Immunol1999;116:360–5.[ISI][Medline]
  31. Goto M, Sasano M, Yamanaka H et al. Spontaneous production of an interleukin-1-like factor by cloned rheumatoid synovial cells in long-term culture. J Clin Invest1987;80:786–96.[ISI][Medline]
  32. Aono H, Hasunuma T, Fujisawa K, Nakajima T, Yamamoto K, Mita S, Nishioka K. Direct suppression of human synovial cell proliferation in vitro by salazosulfapyridine and bucillamine. J Rheumatol1996;23:65–70.[ISI][Medline]
  33. Koch AE. Angiogenesis. Arthritis Rheum1998;41:951–61.[ISI][Medline]
  34. Mottonen T, Paimela L, Ahonen J, Helve T, Hannonen P, Leirisalo-Pero M. Outcome in patients with rheumatoid arthritis treated according to the ‘sawtooth’ strategy. Arthritis Rheum1996;39:996–1005.[ISI][Medline]
  35. Borg G, Allander E, Brodin U, Trang L. Auranofin treatment in early rheumatoid arthritis may postpone early retirement: results from a 2-year double blind trial. J Rheumatol1991;18:1015–20.[ISI][Medline]
  36. Luukkainen R, Kajander A, Isomaki H. Effect of gold on progression of erosions in rheumatoid arthritis: better results with early treatment. Scand J Rheumatol1977;6:189–92.[ISI][Medline]
  37. Morgan SL, Baggott JE, Alarcon GS. Methotrexate and sulfasalazine combination therapy: Is it worth the risk? Arthritis Rheum1993;36:281–2.[ISI][Medline]
  38. Crilly A, McInness IB, McDonald AG, Watson J, Capell HA, Madhok R. Interleukin-6 (IL-6) and soluble IL-2 receptor levels in patients with rheumatoid arthritis treated with low dose oral methotrexate. J Rheumatol1995;22:224–26.[ISI][Medline]
  39. Nagashima M, Yoshino S, Gunji N, Suzuki N, Aono H, Sasano M. Effect of DMARDs on IL-6, bFGF and VEGF in cultured synovial cells—basic research on combination therapy. Arthritis Rheum1998;41:S161.
  40. Cronstein BN, Naime D, Firestein GS, The antiinflammatory effects of an adenosine kinase inhibitor are mediated by adenosine. Arthritis Rheum1995;38:1040–5.[ISI][Medline]
  41. Sajjadi FG, Takabayashi K, Foster AC, Domingo RC, Firestein GS, Inhibition of TNF-{alpha} expression by adenosine: role of A3 adenosine receptors. J Immunol1996;156:3435–42.[Abstract]
  42. Cronstein BN. Molecular therapeutics: Methotrexate and its mechanism of action. Arthritis Rheum1996;39:1951–60.[ISI][Medline]
  43. Paulus HE. The use of combinations of disease-modifying antirheumatic agents in rheumatoid arthritis. Arthritis Rheum1990;33:113–20.[ISI][Medline]
  44. Houssiau FA, Devogelaer J-P, Van Damme J, de Deuxchaisnes CN, Van Snick J. Interleukin-6 in synovial fluid and serum of patients with rheumatoid arthritis and other inflammatory arthritis. Arthritis Rheum1988;31:784–8.[ISI][Medline]
  45. Cohick CB, Furst DE, Quagliata S et al. Analysis of elevated serum interleukin-6 levels in rheumatoid arthritis: Correlation with erythrocyte sedimentation rate or C-reactive protein. J Lab Clin Med1994;123:721–7.[ISI][Medline]
  46. Paleolog EM, Young S, Stark AC, McCloskey RV, Feldmann M, Maini RN. Modulation of angiogenic vascular endothelial growth factor by tumor necrosis factor {alpha} and interleukin-1 in rheumatoid arthritis. Arthritis Rheum1998;41:1258–65.[ISI][Medline]
  47. Nagashima M, Asano G, Yoshino S. Imbalance in production between vascular endothelial growth factor and endostatin exists in patients with rheumatoid arthritis. J Rheumatol2000;27:2339–42.[ISI][Medline]
Submitted 23 February 2000; revised version accepted 9 June 2000.