Cyclosporin A and FK-506 Inhibit Development of Superantigen- Potentiated Collagen-Induced Arthritis in Mice

ISSN 0306-3623/98 $19.00 1 .00 PII S0306-3623(97)00331-5 All rights reserved

Gen. Pharmac. Vol. 30, No. 5, pp. 777–782, 1998 Copyright  1998 Elsevier Science Inc. Printed in the USA.

Cyclosporin A and FK-506 Inhibit Development of SuperantigenPotentiated Collagen-Induced Arthritis in Mice Yuko Takaoka,1* Hiroichi Nagai,1 Masahiko Tanahashi2 and Kenji Kawada3 1 Department of Pharmacology, Gifu Pharmaceutical University, 5-6-1 Mitahorahigashi, Gifu 502, Japan [Tel: 181-58-237-3931; Fax: 181-58-237-5979]; 2 Toray Industries, Inc., Basic Research Laboratories, Kanagawa, Japan; and 3Department of Pathology, Gifu College of Medical Technology, Seki, Japan

ABSTRACT. 1. Staphylococcal enterotoxine B (SEB; superantigen) accelerated the onset of arthritis in mice preimmunized with type II collagen (SEB-potentiated collagen-induced arthritis). Cyclosporin A and FK-506 inhibited the induction and development of clinical signs and histopathological changes of SEB-potentiated collagen-induced arthritis in mice. 2. Simultaneously, both cyclosporin A and FK-506 inhibited the development of humoral and cellular immunity to type II collagen. 3. The expression of IL-2 receptor (CD25) by SEB on splenocyte T cells from collagen-preimmunized mice was inhibited by both agents in ex vivo experimentation. gen pharmac 30;5:777–782, 1998.  1998 Elsevier Science Inc. KEY WORDS. T Cell, CD25, superantigen, cyclosporin A, FK-506 INTRODUCTION Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation within the joints and is associated with synovitis and erosion of cartilage and bone. However, much evidence has been found for the participation of T cells in the pathogenesis of the disease. Many reports describe the infiltration of activated CD41 T cells into the synovial tissue in RA patients (Duke et al., 1982; Kurasaka and Ziff, 1983; Pitzalis et al., 1987; Stamenkovic et al., 1988; Van Boxel and Paget, 1975) and an increase in the quantity of soluble IL-2 receptor in the serum and synovial fluid of these patients (Keystone et al., 1988; Rubin et al., 1990). In addition, recent studies have indicated that the suppression of T cells by irradiation of lymphoid tissue (Brahn et al., 1984; Tannery et al., 1987) and administration of monoclonal antibodies to T cells (anti-CD4 antibody, anti–IL-2R antibody) (Choy et al., 1992; Christian et al., 1989; Daniele et al., 1991; Gerd et al., 1991; John et al., 1992; Kirkhan et al., 1991; Wendling et al., 1992) and drugs such an improvement in refractory RA symptoms. Because of their relationship to T cells, the participation of superantigens in the onset of RA has been investigated extensively. A significant increase in the number of T cells expressing the Vb gene segment in the synovial fluid of RA patients has also been reported (Goronzy et al., 1994; Howell et al., 1991; Jenkins et al., 1993; Paliard et al., 1991; Sioud et al., 1991; Williams et al., 1992). Because superantigens selectively activate T cells through binding to the Vb gene segment, superantigens participate in the onset of RA through the activation of T cells. In addition, it has been reported that superantigens enhance the expression of collagen receptor and antibody-binding sites in the joint (Bret et al., 1994). Such evidence suggests that superantigens are involved in the development of RA. We have previously reported a model of arthritis induced by su*To whom correspondence should be addressed. Received 30 December 1996.

perantigen (SEB) in mice preimmunized with type II collagen (SEBpotentiated collagen-induced arthritis ) (Nagai et al., 1994). In the present study, we examine the effect of cyclosporin A and FK-506 on the development and primary role of T cells in this model. MATERIALS AND METHODS

Animals Male DBA/1J mice, weighing 20–30 g, were used. Mice were obtained from Seiwa Experimental Animals (Fukuoka, Japan). They were housed in plastic cages in an air-conditioned room at 248C, fed a standard laboratory diet and given water ad libitum.

Antigen and superantigen Bovine type II collagen and superantigen, staphylococcal enterotoxine B (SEB; Staphylococcus aureus), were purchased from Cosmobio (Tokyo, Japan) and Serva Feinbiochemica Gumbo & Co. (Germany), respectively.

Drugs Cyclosporin A and FK-506 were donated by Sandoz Ltd. (Basel, Switzerland) and Fujisawa Pharmaceutical Co. Ltd. (Osaka, Japan). Cyclosporin A was dissolved in neutral Miglyol 812 (Hu¨ls Japan, Tokyo, Japan). FK-506 was suspended in 0.5% carboxylmethylcellulose solution. In each experiment, the drug was administered orally six times per week for 9 weeks.

SEB-potentiated collagen-induced arthritis SEB-potentiated collagen-induced arthritis was induced in DBA/1J mice according to the method previously described (Nagai et al., 1994). Eight-week-old male DBA/1J mice were immunized by the intradermal injection of 200 mg of bovine type II collagen suspended in 0.01 M acetic acid and emulsified in complete Freund’s adjuvant

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FIGURE 1. Effect of cyclosporin A (CsA) and FK-506 on staphylococcal enterotoxin B (SEB)-potentiated collagen-induced arthritis in DBA/1J mice as measured by arthritis index. Each drug was administered p.o. six times per week for 9 weeks. (A) Each group consists of five animals. Empty circles: control; filled circles: cyclosporin A (5 mg/kg); empty squares: cyclosporin A (10 mg/kg). (B) Each group consists of six animals. Empty circles: control; filled circles: FK-506 (1 mg/kg); empty squares: FK-506 (3 mg/kg). Each point indicates the mean6SE for arthritis index. Statistical significance in comparison to control at *P,0.05 and **P,0.01, respectively.

(Nacali Tesque Inc., Kyoto, Japan) at the base of the tail. Three weeks after primary immunization, mice were injected with 30 mg of SEB in complete Freund’s adjuvant, intradermally, at the base of the tail. Evaluation of clinical arthritis symptoms was carried out at 3, 4, 5, 6, 7, 8 and 9 weeks after the primary immunization. The degree of arthritis in the extremities of these animals was evaluated clinically by the same observer. The severity of arthritis in the meta-

carpophalangeal wrist, metatarsophalangeal and ankle joints was scored as: 05no arthritis; 15small arthritis; 25severe swelling and non-weight-bearing. The joints score was the sum of scores of all involved joints. The arthritis index was calculated by averaging the total scores. In addition to the arthritis index, the paw volumes were measured by a plethysmometer (TK-101, Unicom). At the end of the experimental period (9 weeks), radiographic assessment of skele-

FIGURE 2. Effect of cyclosporin A (CsA) and FK-506 on staphylococcal enterotoxin B (SEB)-potentiated collagen-induced arthritis in DBA/1J mice (bone destruction score). Each drug was administered p.o six times per week for 9 weeks. (A) Each group consists of five animals. (B) Each group consists of six animals. Each point represents an individual bone destruction score. **Statistical significance in comparison to control at P,0.01.

Inhibition of Collagen-Induced Arthritis

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TABLE 1. Effect of cyclosporin A and FK-506 on superantigen-potentiated collagen-induced arthritis in DBA/1J mice Cyclosporin A group (n 5 5)

FK-506 group (n 5 6)

Organ finding

Gradea

Control

5 mg/kg

10 mg/kg

Control

1 mg/kg

3 mg/kg

Proliferation of synovium

2 1 11 111

0 3 1 1

0 2 2 1

3 2b 0 0

1 0 3 2

4 2b 0 0

3 3b 0 0

Formation of pannus

2 1 11 111

1 2 2 0

0 3 1 1

4 1 0 0

1 0 4 1

5 1b 0 0

6 0c 0 0

Infiltration of cells

2 1 11 111

0 3 1 1

0 2 2 1

3 2b 0 0

1 0 4 1

3 2 1 0

3 3b 0 0

Each drug was administered p.o. at various doses six times/week for 9 weeks. a Grades: 2, negative; 1, slight; 1 1, mild; 1 1 1, marked. b,c Statistical significance in comparison to control at P , 0.05 and P , 0.01, respectively.

tal changes was performed using a Softex x-ray machine. Bone changes were graded on scale of 0–2: 05negative; 15subtle small erosions; 25obvious large erosions in multiple joints. The values for the bone destruction score are equal to the sum of each value from all four joints of the hind-limbs (final total value is scored 0 to 8).

Anti–type II collagen antibody titer An enzyme-linked immunoassay system (ELISA) was employed for the amount of antibodies to bovine type II collagen. This was performed according to the procedure described by Trentman and Dynesius-Trenthman (1983). Briefly, an immunoplate was first coated with 0.5 mg of bovine type II collagen diluted in 50 mM bicarbonate buffer (pH 9.6) and incubated overnight at 48C. The collagen-coated plates were washed with PBS containing 0.05% Tween-20 (PBS-Tween), and blocking with PBS containing 1% bovine serum albumin (1% BSA-PBS) for 2 hr at 378C. After washing with PBS-Tween, the serum samples, which had been diluted at 1:100,000 in 1% BSA-PBS, were then added, and incubated for 2 hr at 378C. After washing, diluted peroxidase-labeled goat antimouse IgG (Cappel, Organon Teknika Co, West Chester, PA, USA) was added to each well. Following an incubation of 2 hr at 378C, the plate was washed with PBS-Tween. A substrate mixture consisting of o-phenylendiamine dihydrochloride (OPD; Sigma) was dissolved in citrate-phosphate before use. The color was allowed to develop for 20 min and the absorbance was read at 492 nm.

Delayed-type hypersensitivity (DTH) response to type II collagen The cell-mediated immune response of mice to type II collagen was estimated as follows: The thickness of both ears of each mouse in the group was measured with a dial thickness gauge (R2-1A, Ozaki Co., Tokyo, Japan). Ten micrograms of type II collagen in phosphate-buffed saline (pH 7.0) was injected into each ear, and the ear thickness of each animal as noted at 24 hr was used as a measurement of in vivo cell-mediated immunity (delayed-type hypersensitivity; DTH) to type II collagen.

Expression of IL-2 receptor (CD25) in SEB-stimulated spleen T cells The effect of drugs on the expression of CD25 in the T cells was examined in ex vivo experiments. Spleens were obtained from mice that had been immunized with type II collagen 3 weeks before. T cells were enriched using a nylon wool column (Wako Pure Chemicals, Osaka, Japan). Enriched T cells (13106) were stimulated by incubation with 10 mg of SEB for 2 hr at 378C in a CO2 humidified incubator. Cells were stained with both fluorescein-isothiocyanateconjugated anti-CD4 and phycoerythrin-conjugated anti-CD25 antibodies (Pharmingen, San Diego, CA) for 30 min on ice in the dark. Then, labeled cells were washed three times and suspended in ice-cold PBS with 0.1% FCS and 0.1% NaN3. Cells were analyzed using a FACScan flow cytometer (Becton-Dickinson, Mountain View, CA).

Statistics Results concerning the arthritis index, bone destruction score, histologic score and the number of CD251CD41 cells were statistically evaluated by Mann–Whitney. Other results were evaluated by Dunnett’s multiple range test. RESULTS

Effect of cyclosporin A and FK-506 on clinical symptoms of SEB-potentiated collagen-induced arthritis The injection of SEB into DBA/1J mice preimmunized with bovine type II collagen resulted in polyarthritis in almost all animals from 5 to 9 weeks after the primary immunization. Treatment with cyclosporin A (10 mg/kg) and FK-506 (1 and 3 mg/kg) inhibited the severity of arthritis, as shown by the arthritis index and bone destruction scores (Figs. 1 and 2). Data on foot-pad volume confirmed the inhibitory effects of the these agents (data not shown). Serial reactions of joints in immunized mice were examined at the more advanced stage and showed marked synovial proliferation, massive infiltration of mononuclear cells and erosion of cartilage with pannus formation. Cyclosporin A (10 mg/kg) and FK-506 (1 or 3 mg/ kg) suppressed each of these histopathological changes (Table 1).

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Y. Takaoka et al. TABLE 2. Effect of cyclosporin A and FK-506 on superantigen-potentiated collagen-induced arthritis in DBA/1J mice Drug Control Cyclosporin A Control FK-506

Dose (mg/kg)

n

Anti–type II collagen (OD) (5 weeks)

Ear swelling (3 1023 cm)

5 10

5 5 5

0.391 6 0.059 0.211 6 0.053 0.081 6 0.012a

30.30 6 0.76 24.07 6 0.50 18.46 6 2.83a

1 3

6 6 6

0.700 6 0.100 0.320 6 0.120a 0.150 6 0.040a

26.85 6 2.48 15.21 6 1.81a 10.85 6 2.07a

Each drug was administered p.o. six times/week for 9 weeks. Blood was taken 5 weeks after the primary immunization. Challenge was carried out nine weeks after the primary immunization with type II collagen. Ear swelling was measured 24 hr after challenge. a Statistical significance in comparison to control at P , 0.01.

The weight of the animals was not affected by immunization and drug treatment (data not shown).

Effect of cyclosporin A and FK-506 on changes of immunological parameters in SEB-potentiated collagen-induced arthritis In SEB-potentiated collagen-induced arthritic mice, the immunological parameters, including the production of IgG antibody to type II collagen and cellular immune response to the antigen, were clearly elevated, after the injection of SEB. Both drugs clearly inhibited the production of anti–type II collagen antibody and onset of the cellular immune response detected by DTH (Table 2).

Effect of cyclosporin A and FK-506 on number of splenocyte T cells expressing IL-2 receptor The number of CD251CD41 cells in 106 enriched T cells from normal mice was almost 104 cells, as shown in Table 3. The number of CD251CD41 T cells increased 4.5 times by immunization with type II collagen when compared with nonimmunization. When preimmunized T cells were stimulated with SEB, the number of CD251CD41 cells increased fourfold before stimulation. Cyclosporin A (10 mg/kg) and FK-506 (1 and 3 mg/kg) significantly inhibited the SEB-induced increase in the number of CD251 cells. DISCUSSION In an earlier report, we described the arthritis model potentiated by superantigen in mice that had been preimmunized with type II col-

lagen (Nagai et al., 1994). These arthritic mice showed clinical symptoms similar to the collagen-induced arthritis model in terms of arthritic severity, bone destruction and histopathological changes. Because the development of this arthritis was inhibited by administration of prednisolone, we concluded that we had evaluated the efficacy of cyclosporin A and FK-506 against the clear suppression of the development of this SEB-potentiated arthritic mice immunized type II collagen. These results also indicated that the inhibitory actions were related to the suppression of the immune response to type II collagen, including the production of humoral antibody, delayedtype hypersensitivity (DTH) reaction and the expression of IL-2 receptor (CD25) on T cells. These results also elucidate the suppressive effect of T-cell immunosuppressors on this type of SEB-potentiated arthritis. In addition to clinical investigation, the role of superantigen in the onset of experimental arthritis in animals has been investigated. Abdelnour et al. (1994) reported that a single injection of superantigen was able to induce arthritis in rats with lower clinical scores and the inoculation of CD41 T cells into the lesion after 48 hr. Wooley and Cingel (1995) reported that pretreatment with superantigen increased the severity of type II collagen-induced arthritis in mice. Kakimoto et al. (1995) reported that a single injection of superantigen produced arthritis in mice and resulted in the production of anti–type II collagen antibody. Our experimental system was different than those of the aforementioned experiments. The purpose of the present study was to research a pharmacological profile for the present system. The results indicated that onset of disease was suppressed due to T-cell immunosuppressors. Cyclosporin A and FK-506 are used to prevent or treat an organ

TABLE 3. Effect of cyclosporin A and FK-506 on number of CD251CD41 cells in splenocytes stimulated with SEB Group 1 2 3 4 5 6 7

Type II collagen

SEB

Drug dose

Number of CD251CD41 cells (3 105 cells) in splenocytes

None Immunized Immunized

None None Stimulated

None None None Cyclosporin A 5 mg/kg Cyclosporin A 10 mg/kg FK-506 1 mg/kg FK-506 3 mg/kg

0.096 6 0.026 0.453 6 0.114 1.692 6 0.225a 1.289 6 0.281 0.321 6 0.083b 0.513 6 0.047b 0.220 6 0.026b

Mice were immunized with type II collagen 3 weeks earlier, except for the normal group. Each drug was administered p.o. six times/week for 3 weeks. Each group consisted of six animals. Each value indicates mean 6 SE of number of CD251CD41 cells. a,b Statistical significance in comparison to group 1 and group 3 at P , 0.01, respectively.

Inhibition of Collagen-Induced Arthritis allograft rejection. Recently, several investigators have reported that these two drugs are effective for the treatment of patients with chronic severe autoimmune disease (Hara et al., 1996; Madhok et al., 1991; Shojania et al., 1995). In experimental animals, there are some reports suggesting the efficacy of these two drugs on type II collagen-induced arthritis and experimental autoimmune encephalomyelitis in mice and rats (Bolton et al., 1992; Kovarik et al., 1995; Inamura et al., 1988; MacComb et al., 1994). Most of the studies reported that the inhibitory mechanisms of these two drugs inhibit the production of antibody and DTH response to type II collagen. In addition, both two drugs inhibit the expression of CD25 on T cells in ex vivo experiments. These results indicate the suppression of T-cell function by the two drugs and suggest the participation of this mechanism in the inhibition of development of disease. In the present experiment, the drugs were administered throughout the entire experimental period. The two drugs may regulate both the primary immune response by antigen and the response stimulated with SEB. Further experiments will be necessary to determine which response is predominantly regulated by the administration of these two agents. At the least, the present results suggest the suppressive effect of cyclosporin A and FK-506 on secondary T-cell activation with SEB. This action is onset of the most important pharmacological profile of these two drugs to explain the suppression of this experimental arthritis. As is well known, mouse T-helper cells are classified into two subsets, T-helper 1 (Th1) cells and T-helper 2 (Th2) cells. Recent studies indicate that superantigens induce relatively high levels of IL-2 and IFN-g production by CD41 T cells, but not IL-4, IL-5 and IL-10 production (Gollob et al., 1993; Maaibe et al., 1995; Nagelerken et al., 1993). In addition, many investigations indicate an important role of Th1 cells in the onset of arthritis (Simon et al., 1994). These data suggest participation of Th1 cells in the development of SEBpotentiated collagen-induced arthritis in mice. The present data, however, do not suggest that any direct evidence of the role of this arthritis will be necessary. In conclusion, the present study demonstrated the efficacy of cyclosporin A and FK-506 on SEB-potentiated collagen-induced arthritis in mice. Both drugs suppressed clinical symptoms of arthritis and the changes in immunological parameters, especially the expression of CD25 on T cells. These present data suggest that these two drugs may have clinical application in arthritis in patients showing a high activation of R cells during the development of the disease. In addition, our study indicated that certain T cells regulated by cyclosporin A and FK-506 play an important role in the onset of SEB-potentiated collagen-induced arthritis in mice. References Abdelnour A., Arvidson S., Bremell T., Holmdahl R. and Tarkowski A. (1994) Host–parasite relationship in experimental Staphylococcus aureus arthritis. Zentralbl. Bakteriol. 26(Suppl.), 493–497. Bolton C. (1992) The efficacy of cyclosporin A, FK-506 and prednisolone to modify the adoptive transfer of experimental allergic encephalomyelitis. Agents Actions 35, 80–84. Brahn E., Helfgott S. M., Belli J. A., Anderson R. J., Peinherz E. L., Schlossman S. F., Austen K. F. and Trenthman D. E. (1984) Total lymphoid irradiation therapy in refractory rheumatoid arthritis; fifteen- to fortymonth follow up. Arthrit. Rheum. 27, 481–488. Bret A. C, Rissing J. P., Thomas B. B., Norma H. B. and Gray K. B. (1994) The effect of growth temperature on Staphylococcus aureus binding to type I collagen. Microbial. Pathogen. 17, 239–251. Choy E. H. S., Chikanza I. C., Kingsley G. H., Corrigall V. and Panayi G. S. (1992) Treatment of rheumatoid arthritis with single dose or weekly pulses of chimeric anti-CD4 monoclonal antibody. Scand. J. Immunol. 36, 291–298. Christian H., Walker C., Muller W., Rieber P., Reiter C., Riethmuller G., Wassmer P., Stockinger H., Madic O. and Werner J. P. (1989) Anti-

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