© Birkhäuser Verlag, Basel, 2004 Inflamm. res. 53 (2004) 469– 474 1023-3830/04/090469-06 DOI 10.1007/s00011-004-1284-y
Inflammation Research
Inflammatory cytokine levels in paw tissues during development of rat collagen-induced arthritis: Effect of FK506, an inhibitor of T cell activation K. Magari, S. Miyata, Y. Ohkubo and S. Mutoh Medicinal Biology Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., 2-1-6, Kashima, Yodogawa-ku, Osaka 532-8514, Japan, Fax: ++81 6 6304 5367, e-mail:
[email protected] Received 4 March 2004; returned for revision 2 April 2004; accepted by M. J. Parnham 9 April 2004
Abstract. Objective and design: To characterize rat collagen-induced arthritis (CIA) on the basis of levels of inflammatory cytokines, tumor necrosis factor (TNF)-a, interleukin (IL)-1b and IL-6 in paw tissues, and further investigate the effect of FK506 (tacrolimus), a potent inhibitor of T cell activation, on cytokine levels. Methods: CIA was induced in female Lewis rats. The volume of hindpaws was measured before and after collagen immunization. TNF-a, IL-1b and IL-6 levels in paw tissue extracts were determined by ELISA. Proteoglycan contents of cartilage in femoral heads was measured as an indication of cartilage destruction. To assess the effect of FK506 on inflammatory cytokine levels, rats were orally treated with 5 mg/kg of FK506 from days 14–21. Results: TNF-a level in paw tissues did not significantly change compared to levels found before collagen immunization, throughout development of CIA. In contrast, IL-1b and IL-6 levels in paw tissues significantly increased between day 14 and day 28 after collagen imuninization, when the arthritis was at a developed stage. Therapeutic treatment with FK506 reduced the elevated level of IL-6, but not IL-1b, in paw tissue. FK506 treatment was effective in suppressing paw swelling and also recovering the loss of proteoglycan contents in the cartilage. Conclusions: Levels of IL-1b and IL-6, but not TNF-a, in paw tissue were upregulated in association with the development of arthritis in rat CIA. These results suggest that IL-1b and IL-6, rather than TNF-a, may play important roles at local inflammatory sites in producing joint destruction in rat CIA. FK506 may improve arthritis in established stages of CIA, by reducing the elevated level of IL-6. Key words: Tumor necrosis factor-a – Interleukin-1b – Interleukin-6 – Rheumatoid arthritis – Collagen-induced arthritis – FK506
Correspondence to: S. Miyata
Introduction Recent studies have revealed key roles for inflammatory cytokines, such as tumor necrosis factor (TNF)-a, interleukin (IL)-1b and IL-6, in pathogenesis of rheumatoid arthritis (RA) [1]. TNF-a, IL-1b and IL-6 are elevated in synovium of RA patients. It has been suggested that those inflammatory cytokines are produced through continuous activation of T cells and interaction of the activated T cells and monocytes/macrophages in RA [2]. Moreover, anti-TNF, IL-1 and IL-6 therapy have been reported to be effective in the treatment of RA [3]. In particular, clinical trials of antiTNF therapy have demonstrated their marked efficacy in controlling signs and symptoms of disease. Thus, it has been postulated that TNF-a plays a pivotal role in the pathogenesis of RA [4]. Collagen-induced arthritis (CIA) is an experimental autoimmune model of human RA that is widely used for studying disease processes, as well as for evaluating possible therapeutic agents. The CIA develops in rodents after immunization with type II collagen. CIA model shares many pathopysiological properties with human RA, such as mononuclear cell infiltration, pannus development, fibrin deposition, cartilage and bone erosion [5, 6]. CIA and human RA have been reported to be T cell-dependent diseases [2, 7]. Anti-TNF [8, 9], anti-IL-1 [10, 11] and anti-IL-6 [12] treatments have also been reported to be effective in suppressing CIA. However, a more recent report showed that TNF knock out mice fully develop arthritis in the CIA model, thus TNF may not be an obligatory cytokine in the pathogenesis of CIA [13]. Quantitative analysis of cytokine levels is essential in study of the role of cytokines in pathogenesis of disease. It has been reported that TNF-a, IL-1b and IL-6 are upregulated during development of CIA, based on analyses of mRNA [14] and immunohistological staining [15, 16]. However, cytokine amounts measured by these methods do not reflect whole protein levels present at inflammatory sites. In the present study, we attempted to characterize the rat CIA model,
470
K. Magari et al.
Inflamm. res.
by quantitating local levels of inflammatory cytokines. We further evaluated the effect of treatment with FK506 (tacrolimus), on inflammatory cytokine levels in established CIA. FK506 is an immunosuppressive agent that specifically suppresses T cell activation by inhibiting calcineurin phosphatase [17–19]. We show here that levels of IL-1b and IL-6, but not TNF-a, in paw tissue are upregulated in association with the development of arthritis in rat CIA, and also suggest that the production of IL1b and IL-6 is regulated differently in this model.
tilled water. The agent was orally administered in a volume of 5 ml/kg. Distilled water was administered as vehicle control. FK506 at a dose of 5 mg/kg was therapeutically administered from days 14–20. Cytokines in paw tissue extracts, paw volume and proteoglycan contents from femoral cartilage were measured on day 21 after drug administration.
Materials and methods
Results
Induction of arthritis
TNF-a, IL-1b and IL-6 levels in paw tissue during development of CIA
Female Lewis rats (7 weeks of age) were obtained from Charles River Japan, Inc. (Kanagawa, Japan) and bred in a clean atmosphere. For immunizations, bovine type II collagen (CII) was dissolved in 0.1 M acetic acid at a concentration of 2 mg/ml and emulsified in an equal volume of incomplete Freund’s adjuvant (Difco Labs, Detroit, MI, USA). Each rat was given intradermal injections of 500 mg CII in an emulsion volume of 500 ml at 2 tail sites and 10 sites on the back on day 0, then received booster injections at 2 tail sites (50 ml in divided doses) on day 7 and day 14. Normal non-immune rats were used as negative controls. The volume of both hind paws was measured on days 0, 7, 14, 17, 21, 24 and 28 by a water displacement method using a plethysmometer for rats. All experimental procedures were reviewed and approved by the Fujisawa Pharmaceutical Animal Experiment Committee.
Determination of TNF-a, IL-1b and IL-6 levels in paw tissue Rats were sacrificed on days 0 (before collagen immunization), 7, 14, 17, 21, 24 and 28. After exsanguination, both hind paws from each rat were dissected above the ankle joint, snap-frozen in liquid nitrogen and stored at –80°C until use. Before homogenization for each assay, frozen paw containing bony tissue was weighed and broken into pieces on dry ice. Paw tissues were added to 4 ml/g tissue of extraction buffer containing 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin and 0.05% Tween 20 in phosphate buffered saline. Tissues were homogenized on ice with a polytron and centrifuged at 5,000 g for 15 min. Supernatants were stored at –80°C until analysis. TNF-a, IL-1b and IL-6 levels in the supernatants were determined using ELISA kits specific for rat TNF-a (Genzyme, Cambridge, MA, USA), IL-1b and IL-6 (both from Endogen, Woburn, MA, USA). The sensitivity of the assays for TNF-a, IL-1b and IL-6 was 5 pg/ml, 12 pg/ml and 15 pg/ml, respectively.
Measurement of Proteoglycan levels After exsanguination, both hindlimbs from each rat were dissected and the heads of femora were separated and stored at –30°C until measurement of proteoglycan levels. Proteoglycan levels in the cartilage of both femoral heads were measured by a method described previously [20], with minor modifications. Briefly, femoral head cartilage was digested for 4 h at 65°C with papain solution (Sigma, St. Louis, MO, USA). Samples from the papain digest were assayed for glycosaminoglycan (GAG) as a measure of proteoglycan content. GAGs were assayed using the 1,9-dimethylmethylene blue binding assay, using chondroitin sulphate (Nacalai Tesque, Kyoto, Japan) as standard.
Drug treatment A solid dispersion formulation of FK506 [21] was prepared at Fujisawa Pharmaceutical Co., Ltd. (Osaka, Japan). FK506 was suspended in dis-
Statistical analysis Results are presented as mean ± SE. t-test was used to compare mean values. P values < 0.05 were considered statistically significant.
Changes in TNF-a levels in paw tissue extracts over the course of CIA were examined as shown in Figure 1. Hind paw volume increased 14 days after primary type-II collagen immunization and remained constant between day 14 and day 28 (Fig. 1A). TNF-a levels in paw tissues of normal nonimmune rats were 0.038 ± 0.023, 0.055 ± 0.010 and 0.020 ± 0.009 ng/g tissue on days 0, 21 and 28, respectively. TNF-a levels in paw tissue from CIA rats were between 0.024 ± 0.004 and 0.064 ± 0.017 ng/g tissue for 28 days after collagen immunization, and did not significantly elevate compared to levels found on day 0 (Fig. 1B). TNF-a level in plasma was also not elevated in CIA (data not shown). IL-1b and IL-6 levels in paw tissue were further examined, as shown in Fig. 2. IL-1b (Fig. 2A) and IL-6 (Fig. 2B) levels in paw tissue of CIA rats significantly increased 4- to 5-fold over levels found before collagen immunization on day 14, and the elevated levels were sustained through to day 28. Effect of therapeutic treatment with FK506 on IL-1b and IL-6 levels in paw tissue of CIA rats An inhibitor of T cell activation, FK506 at a dose of 5 mg/kg, was therapeutically administered to rats from days 14–20. IL-1b and IL-6 levels in paw tissue were determined on day 21. FK506 did not affect IL-1b level in arthritic paws (Fig. 3A). In contrast, the elevated level of IL-6 in arthritic paws was significantly reduced from 3.09 ± 0.42 ng/g tissue in the vehicle treated control animals to 1.10 ± 0.09 ng/g tissue in animals treated with FK506 (Fig. 3B). Effect of therapeutic treatment with FK506 on arthritis of CIA rats FK506 at a dose of 5 mg/kg, was therapeutically administered to rats from days 14–20. Paw swelling and proteogycan content in femoral cartilage were measured as indicators of inflammation and cartilage destruction, respectively, on day 21. Paw swelling was suppressed from 0.61 ± 0.03 ml in the vehicle treated control rats to 0.41 ± 0.05 ml in rats treated with FK506 (Fig. 4A). Proteoglycan levels in the cartilage significantly decreased from 188.4 ± 3.8 mg/cartilage in the normal rats to 157.0 ± 2.7 mg/cartilage in vehicle treated CIA
Vol. 53, 2004
Inflammatory cytokines in rat collagen-induced arthritis
471
Fig. 1. Changes in paw volume and TNF-a level after collagen immunization in CIA. (A) Paw volume before (day 0) and after collagen immunization. (B) TNF-a level in paw tissue extracts before (day 0) and after collagen immunization. Cytokine level was determined by ELISA. Data represent mean ± SE of 5 animals per group. ** P < 0.01 compared to before collagen immunization (day 0).
Fig. 2. Elevation of IL-1b and IL-6 levels during development of CIA. IL-1b (A) and IL-6 (B) levels in paw tissue extracts before (day 0) and after collagen immunization. Cytokine levels were determined by ELISA. Data represent mean ± SE of 5 animals per group. * P < 0.05, ** P < 0.01 compared to before collagen immunization (day 0).
control rats. The proteogycan loss in femoral cartilage was significantly restored (178.2 ± 8.0 mg/cartilage) by the treatment with FK506 (Fig. 4B). Discussion CIA and adjuvant-induced arthritis (AIA) have been widely used as standard models of human RA [6, 22]. It has been reported that levels of TNF-a, IL-1b and IL-6 in ankle joints [23, 24] or paw tissue [25] elevate over the course of rat AIA.
We show here that levels of IL-1b and IL-6, but not TNF-a, in paw tissue are upregulated in association with the development of arthritis in rat CIA. The present study contradicts previous reports on TNF-a expression in CIA. It has been reported that mRNA levels for each of the cytokines TNF-a, IL-1b and IL-6 in arthritic paws increase during the course of CIA [14]. However, cytokine mRNAs do not always correlate with protein levels. These inflammatory cytokines have been detected at protein level using immunohistological methods [15, 16]. We also detected increases in TNF-a positive cells in joint of CIA rats by immunohistochemical analysis [26].
472
K. Magari et al.
Inflamm. res.
Fig. 3. Effect of therapeutic treatment with FK506 on IL-1b (A) and IL-6 (B) levels in paw tissue. Arthritic rats were orally administered FK506 (5 mg/kg) or distilled water (vehicle control) from days 14–20 after collagen immunization. Paw tissues were dissected on day 21. Cytokine levels in the paw extracts were determined by ELISA. Data represent mean ± SE of 5 animals per group. ** P < 0.01 compared to the vehicle control.
Cytokines detected by immunohistological staining may reflect only a small fraction of cytokines as a whole, since soluble cytokines that are abundant in synovial fluids can not be quantitatively measured using immunohistological methods. It is thought that measurement of cytokine levels in paw tissue extracts more precisely reflects whole cytokine levels. However, the fact that TNF-a expression is not measurable in whole joints, does not necessarily negate the potential role of this mediator, as many important mediators may be produced by select tissues in small quantities and act locally. Nishikawa et al. also reported significant increases in levels of IL-1b, but not in TNF-a, in whole joint extracts of CIA rats [27]. In contrast, peripheral blood levels of TNF-a [27, 28] and IL-1b [27] have been reported to increase in rat CIA. It is not clear whether peripheral blood levels of these cytokines are associated with the disease progression, as TNF-a and IL-1b were analyzed only at the end of the disease. We also attempted to compare TNF-a, IL-1b and IL-6 levels in paw tissue and plasma in CIA. However, significant elevation of these cytokines in plasma was not observed during development of CIA. The analysis of IL-1b and IL-6 levels in whole paw extracts may be useful for studying the roles of the cytokines in CIA, as the kinetics appear to be associated with the development of arthritis. It has been suggested that inflammatory cytokines are produced through continuous activation of T cells and interaction of the activated T cells and monocytes/macrophages in RA [2]. CIA as well as human RA have been reported to be T cell-dependent diseases [2, 7]. To investigate whether IL1b and IL-6 are produced through T cell activation, the effect of an inhibitor of T cell activation, FK506, was studied. FK506 exerts its immunosuppressive effects by inhibiting calcineurin phosphatase, an enzyme involved in activation of transcription factor NF-AT, required for the expression of
Fig. 4. Effect of therapeutic treatment with FK506 on paw swelling (A) and cartilage destruction of femoral heads (B) in CIA. Arthritic rats were orally administered FK506 (5 mg/ kg) or distilled water (vehicle control) from days 14-20 after collagen immunization. Paw volume and proteoglycan levels in femoral heads were measured on day 21. Data represent mean ± SE of 5 animals per group. ## P < 0.01 compared to normal non-immune rats. * P < 0.05, ** P < 0.01 compared to the vehicle control.
cytokine genes in T cells [19]. FK506 is reported to suppress the onset of CIA in prophylactic treatment [26, 29], and is also effective in the treatment of RA [30]. We have shown that FK506 reduces both levels of IL-1b [25, 31] and IL-6 [25] in paw tissue during therapeutic treatment of AIA. As shown in the present study, FK506 reduced elevated level of IL-6, but not IL-1b, in arthritic paws of CIA rats. It has been shown that both T cells and monocytes/macrophage produce IL-6, although T cells do not produce IL-1b. FK506 inhibits IL-1b and IL-6 production triggered by T cell activation, but does not affect production of these cytokine induced by stimulants, such as lipopolysaccharide, which directly activate monocytes/macrophages [32, 33]. Thus, differential effects of FK506 on IL-1b and IL-6 levels suggest that IL-6, but not IL-1b, may be produced through a T cell activation-dependent mechanism in CIA. Therapeutic treatment with FK506 was effective in suppressing paw swelling and restoring proteoglycan loss in car-
Vol. 53, 2004
Inflammatory cytokines in rat collagen-induced arthritis
tilage. Anti-IL-6 receptor treatment has been shown to suppress arthritis progression in CIA [12]. It is therefore possible that selective reduction of the level of IL-6 by treatment with FK506 leads to suppression of arthritis in established CIA. Anti-TNF therapy has been focused in the treatment of RA, based on marked efficacy in the treatment of RA [4]. Adenovirus-mediated transfer of a modified TNF-a receptor gene suppresses arthritis in rat CIA [34]. Anti-TNF treatments have also been reported to be effective in suppressing CIA. However, some of those treatments do not appear to produce potent suppressive effects on CIA [35, 36]. More recently, it has been shown that TNF knock out mice fully develop arthritis in the CIA model, so that arthritis can develop through TNF-independent mechanisms in CIA [13]. In addition to the study based on anti-cytokine treatment or cytokine gene manipulation, quantitative analysis of cytokine levels provides much insight into the role of cytokines in disease pathogenesis. It is notable that TNF-a levels in paw tissue do not change throughout the course of rat CIA, although TNF-a levels in paw tissues are elevated significantly more than 3-fold over levels before arthritis induction in AIA [25]. TNF-a, IL-1b and IL-6 possess many overlapping actions, such as induction of adhesion molecules, matrix metalloproteinase and prostaglandin E2, plus osteoclast activation [37, 38]. The present results raise the possibility that IL-6 and IL-1b rather than TNF-a, may play important roles at the level of local inflammation sites in producing joint destruction in CIA. References [1] Feldmann M, Brennan FM, Maini RN. Role of cytokines in rheumatoid arthritis. Annu Rev Immunol 1996; 14: 397–440. [2] Panayi GS, Lanchbury JS, Kingsley GH. The importance of the T cell in initiating and maintaining the chronic synovitis of rheumatoid arthritis. Arthritis Rheum 1992; 35: 729–35. [3] Maini RN, Taylor PC. Anti-cytokine therapy for rheumatoid arthritis. Annu Rev Med 2000; 51: 207–29. [4] Feldmann M, Maini RN. Anti-TNFa therapy of rheumatoid arthritis: What have we learned? Annu Rev Immunol 2001; 19: 163–96. [5] Trentham DE, Townes AS, Kang AH. Autoimmunity to type II collagen: an experimental model of arthritis. J Exp Med 1977; 146: 857–68. [6] Durie FH, Fava RA, Noelle RJ. Collagen-induced arthritis as a model of rheumatoid arthritis. Clin Immunol Immunopathol 1994; 73: 11–8. [7] Klareskog L, Holmdahl R, Larsson E, Wigzell H. Role of T lymphocytes in collagen II induced arthritis in rats. Clin Exp Immunol 1983; 51: 117–25. [8] Williams RO, Feldmann M, Maini RN. Anti-tumor necrosis factor ameliorates joint disease in murine collagen-induced arthritis. Proc Natl Acad Sci USA 1992; 89: 9784–8. [9] Bendele AM, Chlipala ES, Scherrer J, Frazier J, Sennello G, Rich WJ et al. Combination benefit of treatment with the cytokine inhibitors interleukin-1 receptor antagonist and PEGylated soluble tumor necrosis factor receptor type I in animal models of rheumatoid arthritis. Arthritis Rheum 2000; 43: 2648–59. [10] van den Berg WB, Joosten LAB, Helsen M, van de Loo FAJ. Amelioration of established murine collagen-induced arthritis with anti-IL-1 treatment. Clin Exp Immunol 1994; 95: 237–43. [11] Bendele A, McAbee T, Sennello G, Frazier J, Chlipala E, McCabe D. Effiicacy of sustained blood levels of interleukin-1 receptor antagonist in animal models of arthritis. Comparison of efficacy in animal models with human clinical data. Arthritis Rheum 1999; 42: 498–506.
473 [12] Takagi N, Mihara M, Moriya Y, Nishimoto N, Yoshizaki K, Kishimoto T, et al. Blockage of interleukin-6 receptor ameliorates joint disease in murine collagen-induced arthritis. Arthritis Rheum 1998; 41; 2117–21. [13] Campbell IK, O’Donnell K, Lawlor KE, Wicks IP. Severe inflammatory arthritis and lymphadenopathy in the absence of TNF. J Clin Invest 2001; 107: 1519–27. [14] Thornton S, Duwel LE, Boivin GP, Ma Y, Hirsch R. Association of the course of collagen-induced arthritis with distinct patterns of cytokine and chemokine messenger RNA expression. Arthritis Rheum 1999; 42: 1109–18. [15] Marinova-Mutafchieva L, Williams RO, Mason LJ, Mauri C, Feldmann M, Maini RN. Dynamics of proinflammatory cytokine expression in joints of mice with collagen-induced arthritis (CIA). Clin Exp Immunol 1997; 107: 507–12. [16] Palmblad K, Erlandsson-Harris H, Tracey KJ, Andersson U. Dynamics of early synovial cytokine expression in rodent collagen-induced arthritis. A therapeutic study using a macrophagedeactivating compound. Am J Pathol 2001; 158: 491–500. [17] Kino T, Hatanaka H, Miyata S, Inamura N, Nishiyama M, Yajima T et al. FK-506, a novel immunosuppressant isolated from a Streptomyces. II. Immunosuppressive effect of FK-506 in vitro. J Antibiotics 1987; 40: 1256–65. [18] Tocci MJ, Matkovich DA, Collier KA, Kwok P, Dumont F, Lin S et al. The immunosuppressant FK506 selectively inhibits expression of early T cell activation genes. J Immunol 1989; 143: 718–26. [19] Ho S, Clipstone N, Timmermann L, Northrop J, Graef I, Fiorentino D et al. The mechanism of action of cyclosporin A and FK506. Clin Immunol Immunopathol 1996; 80: S40–5. [20] Karran EH, Young TJ, Markwell RE, Harper GP. In vivo model of cartilage degradation – effects of a matrix metalloproteinase inhibitor. Ann Rheum Dis 1995; 54: 662–9. [21] Honbo T, Kobayashi M, Hane K, Hata T, Ueda Y. The oral dosage form of FK-506. Transplant Proc 1987;19 Suppl 6: 17–22. [22] Billingham MEJ. Models of arthritis and the search for antiarthritic drugs. Pharmac Ther 1983; 21: 389–428. [23] Smith-Oliver T, Noel LS, Stimpson SS, Yarnall DP, Connolly KM. Elevated levels of TNF in the joints of adjuvant arthritic rats. Cytokine 1993; 5; 298–304. [24] Szekanecz Z, Halloran MM, Volin MV, Woods JM, Strieter RM, Haines III GK et al. Temporal expression of inflammatory cytokines and chemokines in rat adjuvant-induced arthritis. Arthritis Rheum 2000; 43: 1266–77. [25] Magari K, Miyata S, Nishigaki F, Ohkubo Y, Mutoh S, Goto T. Differential effects of FK506 and Methotrexate on inflammatory cytokine levels in rat adjuvant-induced arthritis. J Rheumatol 2003; 30: 2193–200. [26] Magari K, Nishigaki F, Sasakawa T, Ogawa T, Miyata S, Ohkubo Y, et al. Anti-arthritic properties of FK506 on collagen-induced arthritis in rats. Inflamm Res 2003; 52: 524–529. [27] Nishikawa N, Myoui A, Tomita T, Takahi K, Nampei A, Yoshikawa H. Prevention of the onset and progression of collagen-induced arthritis in rats by the potent p38 mitogen-activated protein kinase inhibitor FR167653. Arthritis Rheum 2003; 48: 2670–81. [28] Campo GM, Avenoso A, Campo S, Ferlazzo AM, Altavilla D, Calatroni A. Efficacy of treatment with glycosaminoglycans on experimental collagen-induced arthritis in rats. Arthritis Res Ther 2003; 5: R122–31. [29] Inamura N, Hashimoto M, Nakahara K, Aoki H, Yamaguchi I, Kohsaka M. Immunosuppressive effect of FK506 on collageninduced arthritis in rats. Clin Immunol Immunopathol 1988; 46: 82–90. [30] Furst DE, Saag K, Fleischmann MR, Sherrer Y, Block JA, Schnitzer T et al. Efficacy of tacrolimus in rheumatoid arthritis patients who have been treated unsuccessfully with methotrexate. A six-month, double-blind, randomized, dose-ranging study. Arthritis Rheum 2002; 46: 2020–8. [31] Magari K, Miyata S, Ohkubo Y, Mutoh S, Goto T. Calcineurin inhibitors exert rapid reduction of inflammatory pain in rat adjuvant-induced arthritis. Br J Pharmacol 2003; 139: 927–34.
474 [32] Sakuma S, Kato Y, Nishigaki F, Sasakawa T, Magari K, Miyata S et al. FK506 potently inhibits T cell activation induced TNF-a and IL-1b production in vitro by human peripheral blood mononuclear cells. Br J Pharmacol 2000; 130: 1655–63. [33] Sakuma S, Kato Y, Nishigaki F, Magari K, Miyata S, Ohkubo Y, et al. Effects of FK506 and other immunosuppressive anti-rheumatic agents on T cell activation mediated IL-6 and IgM production in vitro. Int Immunopharmacol 2001; 1: 749–57. [34] Le CH, Nicolson AG, Morales A, Sewell KL. Suppression of collagen-induced arthritis through adenovirus-mediated transfer of a modified tumor necrosis factor a receptor gene. Arthritis Rheum 1997; 40: 1662–9. [35] Joosten LAB, Helsen MMA, van de Loo FAJ, van den Berg WB. Anticytokine treatment of established type II collagen-induced
K. Magari et al.
Inflamm. res.
arthritis in DBA/1 mice. A comparative study using anti-TNFa, anti-IL-1a/b, and IL-1Ra. Arthritis Rheum 1996; 39: 797–809. [36] Joosten LAB, Helsen MMA, Saxne T, van de Loo FAJ, Heinegård D, van den Berg WB. IL-1ab blockade prevents cartilage and bone destruction in murine type II collagen-induced arthritis, whereas TNF-a blockade only ameliorates joint inflammation. J Immunol 1999; 163: 5049–55. [37] Ridderstad A, Abedi-Valugerdi M, Moller E. Cytokines in rheumatoid arthritis. Ann Med 1991; 23: 219–23. [38] Manolagas SC. Role of cytokines in bone resorption. Bone 1995; 17 Suppl 2: 63S–7S.
