Agents Actions 41, Special Conference Issue: C267-C270 (1994)
0065-4299/94/04C267-04 $1.50 +0.20/0 9 1994 Birkh/iuser Verlag, Basel
14. Leflunomide Suppression of autoimmune responses and inflammatory events by leflunomide in an animal model for rheumatoid arthritis T. T. Giant 1'2, K. Mikecz 1'2, F. Brennan 1, G. Negroiu 1, and R. R. Bartlett 3 1 Departments of Biochemistry and 2 Orthopedic Surgery, Rush Medical University at Rush-Presbyterian-St Luke's Medical Center, 1653 West Congress Parkway, Chicago, IL 60612, USA 3 Pharmakologische Forschung, Hoechst AG, Werk Kalle-Albert, D-65174 Wiesbaden, Germany
Abstract. The effect ofleflunomide (HWA 486) was tested in proteoglycan-induced arthritis in an autoimmune animal model showing many similarities to human rheumatoid arthritis and ankylosing spondylitis. The development of the disease in genetically susceptible BALB/c mice is dependent upon the expression of both cell-mediated and humoral immunity to host mouse cartilage proteoglycan. Arthritic and control (non-arthritic) animals were treated with 35 mg leflunomide/kg body weight/day for 12 weeks to suppress inflammatory events and antibody titers. Leflunomide suppressed acute inflammatory events, protected animals from new inflammatory episodes and acute exacerbations, slightly reduced the stiffness in joints and blocked the degradation of cartilage. The suppressive effect of leflunomide in proteoglycan-induced arthritis is due primarily to the suppression of autoantibody formation.
zing fetal human proteoglycan and cross-react with both native and degraded proteoglycans of mouse cartilage [2, 6, 7] and an antibody-dependent T cell-mediated reaction creates the pathological basis of the organ-specific inflammatory process [-8, 9]. In this study we used leftunomide (HWA 486), an isoxazol derivative [10, 11]. to suppress/modify the immune responses and inflammatory events in proteoglycan-induced arthritis. We selected arthritic animals from a large pool immunized BALB/c mice having the same (auto)antibody levels and clinical scores of arthritis. These animals were divided into experimental (treated) and control (untreated) groups to monitor the effect of leflunomide.
Introduction
Antigens and induction of primary arthritis
Immunization of BALB/c mice with fetal human proteoglycan depleted of chondroitin sulfate produces progressive polyarthritis and ankylosing spondylitis [,1-4]. The initial external symptoms of joint inflammation (swelling and redness) appear after the 3rd-5th antigen injection depending upon the BALB/c colony and the proteoglycan preparation used. This mouse model shows many similarities to human rheumatoid arthritis and ankylosing spondylitis as indicated by clinical assessments and histopathologic studies of diarthrodial joints and spine [-1, 3, 5]. The arthritis often starts as a bilateral, polyarticular synovitis in small peripheral joints and becomes progressive, with extensive erosion of cartilage and bone within the joint leading to joint deformities and ankylosis. The development of the disease in BALB/c mice is dependent upon the expression of both cell-mediated and humoral immunity to host mouse cartilage proteoglycan [-2-4]. Autoantibodies and T cells react with the immuni-
High buoyant density cartilage proteoglycans (aggrecans) were prepared from newborn human articular cartilage as well as from 1-week old mouse epiphyseal cartilage as described [1]. Animals were sensitized by intraperitoneal injection of 100 gg of chondroitinase ABC-treated proteoglycan (measured as protein) in 100 gl of phosphate-buffered saline (PBS), pH 7.2, and Freund's complete adjuvant (in a 1 : 1 emulsion with PBS). They were reinjected twice more with the antigen in Freund's incomplete adjuvant at 1 and 3 weeks. All BALB/c mice immunized with human proteoglycan developed arthritis in diarthrodial joints after the third antigen injection. Animals were tested for antibodies during weeks 12-18 of the immunization. Sera from BALB/c mice with progressive polyarthritis were tested for antibodies to fetal/newborn human proteoglycan and for autoantibodies to intact and degraded (chondroitinase ABC-treated) mouse proteoglycans using radioimmunoassay as described [1, 2]. Control female BALB/c mice were immunized with non-arthritogenic (bovine articular) proteoglycan or injected with saline and adjuvant with proteoglycan antigens.
Correspondence to: T. T. Glant
Materials and methods
Experimental groups Fifty-two arthritic BALB/c mice were used in this experiment to monitor simultaneously both chronic and acute inflammatory
Agents Actions, Special Conference Issue (1994)
C268 Antibodies to native mouse proteoglycan 20
~5000 ~4000
joints. For example, a maximum of grade score 8 was given for one limb, if all joints were heavily inflamed. Chronicity of arthritis was characterized by measuring the loss of extension in the ankle joint [5, 13]. Limbs, tails and lumbar spine were fixed, decalcified and embedded in paraffin. Sections were stained with hematoxylin and eosin and toluidine blue [1, 6, 13].
Statistical analysis
~3000 5 ~2000
41000 4
8
12
16
23
10
40
61
83
102
123
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DAYS OF EXPERIMENT
~ Antibodies to degraded mouse proteoglycan
Results 20
~5000
~ 4000 xo
4 5000 "~2000 1000
8
12
16
23
30
40
61
83
102
123
Clinical scores were recorded three times a week by two independent investigators. Antibody titers were measured in triplicate samples by radioimmunoassay [1, 2]. Mean values were used for calculation. Non-parametric statistics were used to analyze results, including Wilcoxon's signed ranks test and Friedman's analysis of variance. Values of p < 0.05 were regarded as statistically significant.
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DAYS OF EXPERIMENT
Fig. 1. Serum antibody levels and cumulative clinical scores in non-treated (A) and leflunomide-treated (B) arthritic animals. Arrows indicate the beginning [$] and end [i"] of treatment with leflunomide (Fig. 1B). Autoantibody levels (at 1:500 serum dilutions) to native and degraded mouse proteoglycans are indicated by columns and clinical scores by lines.
responses as well as antibody titers in leflunomide-treated and non-treated arthritic groups. Animals immunized with non-arthritogenic proteoglycan from bovine articular cartilage and nonimmunized (saline and adjuvant-injected) female BALB/c mice served as controls. Treated animals (arthritic, non-arthritic and nonimmunized) were fed daily (Monday Friday) with 35 mg/kg body weight/day leflunomide suspended in carboxymethylcellulose (Serva, Heidelberg; 1%, w/v in water) as described [12]. The body weight was measured daily. Untreated control animals (arthritic, immunized with non-arthritogenic proteoglycans and non-immunized) were fed only carboxymethylcellulose without leflunomide. The first "per os" treatment with or without leflunomide is indicated as day 1 (Fig. 1). All animals were treated with leflunomide for 12 weeks and were sacrificed 7 weeks later.
Assessment of disease activity The appendages of all mice (leflunomide-treated and non-treated: immunized with arthritic or non-arthritic proteoglycans and nonimmunized animals) were examined three times a week to record clinical arthritic changes and were documented as described [1, 5, 13]. Swelling measured as thickness of wrist, elbow, knee and intermalleolar diameters, redness and loss of extension of ankle joints (normally 180~ were recorded. An.acute clinical score, which is the cumulative score of the four limbs of each animal at each time (three times a week), were determined. Each paw was scored from 0 to 8, where no redness or swelling was recorded as 0. Acute clinical score was graded as redness of paws (score 1) and/or swelling (mild, moderate and severe: scores 1-3) of the wrist, elbow, ankle and knee
Non-treated arthritic animals N o n - t r e a t e d arthritic animals showed characteristic progressive arthritic changes as previously described [1, 3, 7, 14]. Clinical s y m p t o m s of acute inflammation appeared and regressed in different peripheral joints of the same animal. I n f l a m m a t o r y episodes in originally acutely inflamed joints led to deformities, ankylosis (Fig. 1A) and complete loss of articular cartilage by the end of the experiment. The a m o u n t of autoreactive antibodies to mouse cartilage proteoglycan did not decrease significantly in the sera of arthritic animals during the entire period of the experiment (Fig. 1A).
Leflunomide-treated arthritic animals These.animals had very severe progressive polyarthritis with b o t h acute and chronic forms of inflammation in peripheral joints at the beginning of the treatment period. The acute inflammation significantly regressed from the second week of leflunomide treatment and was hardly visible after the third or fourth week of the experiment (Fig. 1B). " S p o n t a n e o u s " exacerbation of mild and transient inflammation was rarely detected during the leflunomide treatment. The joint stiffness and deformities and reduced extension (expressed as chronic clinical score) did not worsen during the treatment period; in fact, it even decreased slightly. However, the decrease of chronic s y m p t o m s was less impressive than the decrease of acute inflammation (Fig. 1B). Mild clinical s y m p t o m s of inflamm a t i o n (edema, redness, swelling and a moderate increase [ 5 - 1 0 % ] in joint diameter), however, returned 2 - 3 weeks after terminating the drug treatment (Fig. 1B). These clinical s y m p t o m s were, however, less intensive than the spontaneous exacerbations observed in non-treated arthritic animals or in these leflunomide-treated arthritic animals prior to treatment. The m o s t i m p o r t a n t histopathological observation was that only m o d e r a t e cartilage d a m a g e was found in the joints of animals treated with leflunomide, while complete deterioration of the articular surface developed at the end of the experiment in arthritic animals not treated with leflunomide. Circulating antibodies against b o t h the immunizing fetal h u m a n cartilage proteoglycan and autoantibodies to
Agents Actions, Special Conference Issue (1994) mouse proteoglycans decreased significantly after the introduction of leflunomide treatment (Fig. 1B).
Leflunomide-treated and non-treated immunized, but non-arthritic, animals
These animals were used as controls to detect serum antibody levels against bovine proteoglycans which were found to be non-arthritogenic. Although high serum antibody levels stagnated in non-arthritic animals during the entire period of the experiment, the concentration of circulating anti-proteoglycan antibodies dropped within 1-2 weeks after the introduction of treatment with leflunomide (data not shown) [4]. This remained at a low level during the treatment. However, approximately 2 weeks after terminating the leflunomide treatment, the antibody titers against immunizing bovine cartilage proteoglycan in the sera of animals increased explosively (data not shown) [4].
Non-immunized, non-arthritic animals treated with leflunomide
These BALB/c mice were injected with an emulsion of physiological saline and Freund's adjuvants without proteoglycan antigens. None of these animals produced antibodies to any cartilage proteoglycans or developed arthritis. Although the leflunomide-treated animals lost body weight (data not shown), no other clinical symptoms appeared in treated animals.
C269 effect of leflunomide is due primarily to the suppression of autoreactive antibodies. Although more detailed and longitudinal experiments are required to identity the exact mechanism for leflunomide in autoimmune arthritides and to study the protective effect of this drug for the development of autoreactive antibodies and inflammatory mediators, this study further confirms that leflunomide is a potential drug for the treatment of patients with seropositive rheumatoid arthritis or systemic lupus erythematosus [15]. Acknowledgements. Authors wish to thank Dr. K/immerer for pro-
viding leflunomide. References [1] T. T. Glant, K. Mikecz, A. Arzoumanian and A. R. Poole, Proteoglycan-induced arthritis in BALB/c mice. Clinical features and histopathology. Arth. Rheum. 30, 201-212 (1987). [-2] K. Mikecz, T. T. Glant and A. R. Poole, Immunity to cartilage proteoglycans in BALB/c mice with progressive polyarthritis and ankylosing spondylitis induced by injection of human cartilage proteoglyean. Arth. Rheum. 30, 306 318 (1987). [-3] K. Mikecz, T. T. Glant, E. Bu2as and A. R. Poole, Cartilage proteoglycans as potential autoantigens in humans and in experimental animals. Agents and Actions 23, 63-66 (1988).
[-4] T. T. Glant, K. Mikecz, R. R. Bartlett, F. Deft.k, E. J.-M. A. Thornar, J. M. Williams, T. Mattar, K. E. Kuettner and R Schleyerbach, Immunomodulation of proteoglycan-induced progressive polyarthritis by leflunomide. Immunopharmacology 23, 105-116 (1992). [-5] J. M. Williams, J. Zurawski, K. Mikecz and T. T. Glant, Functional assessment of joint use in experimental inflammatory murine arthritis. J. Orthop. Res. 11, 172-180 (1993).
[-6] E. Dayer, L. Mathai, T. T. Glant, K. Mikecz and A. R. Poole, Discussion Leflunomide is a novel antiphlogistic and immunomodulating agent that has been shown to be efficacious in preventing and healing autoimmune disorders and reactions leading to organ transplantation rejection [15-17]. B cells are especially susceptible to this agent, i.e., inhibiting cell proliferation and suppressing their antibody formation as well as interfering in the function of certain T cell products necessary for B cell differentiation [10, 15]. Leflunomide inhibits tyrosine kinase [-15, 18], most likely acting directly upon the EGF-receptor tyrosine kinase. This may explain the non-cytot0xic and reversible antiproliferative activity upon various cells, including T cell proliferation [18] and the proliferation and differentiation of B cells to plasma cells. Although leflunomide antagonizes the effects of many cytokines [15, 18] the expression and synthesis o'f interleukin-1 is not antagonized by this drug [15]. On the other hand, leflunomide inhibits the release of certain metabolites of arachidonic acid and histamine which may offer additional anti-inflammatory effects of this agent [15, 19]. In our experiments, leflunomide suppressed acute inflammatory events in proteoglycan-induced arthritis and blocked the progression of chronic arthritis in previously involved joints. Since the regression of inflammatory events and the decline of antibody levels showed a strong correlation during drug treatment, we believe that the
Cartilage proteoglycan-indueed arthritis in Balb/c: Antibodies recognize human and mouse cartilage proteoglycan and can cause depletion of cartilage proteoglycan with little or no synovitis. Arth. Rheum. 33, 1394-1405 (1990).
[-7] T. T. Glant, F. Ffil6p, K. Mikecz, E. Buzfis, G. Molnfir and P. Erhardt, Proteoglycan-specific autoreaetive antibodies and Tlymphocytes in experimental arthritis and human rheumatoid joint diseases. Biochem. Soc. Trans. 18, 796-799 (1990). [-8] K. Mikecz, T. T. Glant, E. Buz/ts and A. R. Poole, Proteoglycan-induced polyarthritis and spondylitis adoptively transferred to naive (nonimmunized) BALB/c mice. Arth. Rheum. 33,
866-876 (1990). [-9] K. Mikecz, and T. T. Glant, Migration and homing oflymphocytes to lymphoid and synovial tissues in proteoglycan-induced murine arthritis. Arth. Rheum. 37, in press (1993). [10] R. R. Bartlett, Immunopharmacological profile of HWA 486, a novel isoxazol derivative IL In vivo immunomodulating effects differ from those of cyclophosphamide, prednisolone, or cyclosporin A. Int. J. Immunopharmacol. 8, 199-204 (1986). [-11] R. R. Bartlett and R. Schleyerbach, Immunopharmacological profile of a novel isoxazol derivative, HWA 486, with potential antirheumatic activity - I. Disease modifying action on adjuvant arthritis of the rat. Int. J. Immunopharmacol. 7, 7-17 (1985). [12] R. R. Bartlett, S. Popovic and R. X. Raiss, Development of autoimmunity in MRL/lpr mice and the effects of drugs on this murine disease. Scand. J. Rheumatol. 75, 290-299 (1988).
[13] E. I. Buzfis,K. Hol16,L. Rubliczky, M. Garz6, P. Nyirkos and T. T. Glant, Effect of pregnancy on proteoglycan-induced progressive polyarthritis in Balb/c mice: Remission of disease activity. Clin. Exp. Immunol. 94, 252-260 (1993).
[14] T. T. Glant, K. Mikecz, E. Buzfis, E. Dayer, and A. R. Poole, Anti-proteoglycan antibodies in experimental spondylitis. In: Monoclonal Antibodies, Cytokines, and Arthritis, Mediators of
C270 Inflammation and Therapy, (Ed. T. Kresina) pp. 341 356, Marcel Dekker, New York 1991. [15] R. R. Bartlett, M. Dimitrijevic, T. Mattar, T. Zielinski, T. German, E. R/ide, G. H. Thoenes, C. C. A. Kfichle, H.-U. Schorlemmer, E. Bremer, A. Finnegar and R. Schleyerbach, Leflunomide (HWA 486), a novel immuno-modulating eompound for the treatment of autoimmune disorders and reactions leading to transplantation rejection. Agents and Actions 32, 10-21 (1991). [16] J. W. Williams, F. Xiao, P. F. Foster, A. Chong, S. Sharma, R. Bartlett and H. S. Sankary, Immunosuppressive effects of leflunomide in a cardiac allograft model. Transplant. Proc. 25, 745-746 (1993). [17] A. S.-F. Chong, H. Gebel, A. Finnegan, E. E. Petraitis, X. L. Jian, H. N. Sankary, P. Foster and J. W. E. Williams, Lef-
Agents Actions, Special Conference Issue (1994) lunomide, a novel immunomodulatory agent: In vitro analyses of the mechanism of immunosuppression. Transplant. Proc. 25, 747-749 (1993). [18] A. S.-F. Chong, A. Finnegan, X. Jiang, H. Gebel, H. N. Sankary, P. Foster and J. W. Williams, Leflunomide, a novel immunosuppressive agent: the mechanism of inhibition of T cell proliferation. Transplant. Proc. 55, 1361-1366 (1993). [19] R. R. Bartlett, T. Mattar, U. Weithmann, H. Anagnostopulos, S. Popovic and R. Schleyerbach, Leflunomide, (HWA 486). A novel immunorestoring drug. In: Therapeutic Approaches to Inflammatory Diseases. (Eds. A. J. Lewis, N. S. Doherty and N. R. Ackerman) pp. 215-228, Else-~ier, New York 1989.
