Humanized Antibody to Human Interleukin-6 Receptor Inhibits the Development of Collagen Arthritis in Cynomolgus Monkeys

Clinical Immunology Vol. 98, No. 3, March, pp. 319 –326, 2001 doi:10.1006/clim.2000.4989, available online at http://www.idealibrary.com on

Humanized Antibody to Human Interleukin-6 Receptor Inhibits the Development of Collagen Arthritis in Cynomolgus Monkeys Masahiko Mihara, Masao Kotoh, Norihiro Nishimoto,* Yasuhiro Oda,† Eiji Kumagai,† Nobuhiro Takagi, Kunihiko Tsunemi, Yoshiyuki Ohsugi, Tadamitsu Kishimoto,‡ Kazuyuki Yoshizaki,* and Yasuhisa Takeda Fuji-Gotemba Research Laboratories, Chugai Pharmaceutical Co. Ltd., Shizuoka, Japan; †CSK Research Park Inc., Nagano, Japan; *Department of Medical Science I, School of Health and Sport Sciences, Osaka University, Osaka, Japan; and ‡Osaka University, Osaka, Japan

In the present study, we demonstrated the anti-arthritic effect of humanized anti-human IL-6 receptor (IL-6R) antibody, MRA, in cynomolgus monkey. MRA can react with monkey IL-6R and block signal transduction of IL-6. Collagen-induced arthritis (CIA) was induced by twice immunizing with bovine type II collagen (CII) emulsified with complete adjuvant. MRA was intravenously injected once a week, from the day of the first collagen immunization, for 13 weeks. The symptoms of arthritis were evaluated using a visual scoring system and radiography. Inflammatory parameters (C-reactive protein (CRP), fibrinogen, and erythrocyte sedimentation rate (ESR) and concentrations of anti-CII antibody, anti-MRA antibody, and MRA were monitored regularly. At the end of the study, histological evaluation was carried out. MRA, at a dose of 10 mg/kg, gave rise to statistically significant suppression. The elevation of serum CRP and fibrinogen levels and ESR were also inhibited. Furthermore, radiographic and histological examination showed that MRA treatment suppressed joint destruction. Our results demonstrate that IL-6 plays an important role in monkey CIA and that MRA may be an attractive agent for the treatment of rheumatoid arthritis. © 2001 Academic Press

Key Words: anti-IL-6R antibody; monkey; collagen arthritis; treatment. INTRODUCTION

Interleukin-6 (IL-6) is a multifunctional cytokine and its biological activities reflect the clinical symptoms of rheumatoid arthritis (RA), i.e., production of acute phase protein and autoantibody, elevation of platelet numbers, induction of fever, and decrease in erythrocyte numbers (1). In addition, elevated production of IL-6 was observed in RA patients (2, 3) and IL-6 levels are closely related to disease activity (4). Previously, we reported that rat anti-mouse IL-6R antibody prevented the development of collagen-induced arthritis (CIA) in DBA/1J mice (5). These lines of evidence strongly suggest that IL-6 plays an essential role in the

pathogenesis of arthritis and encouraged us to develop anti-human IL-6R antibody as an anti-arthritic agent. CIA is inducible in nonhuman primates (6 – 8). The study of arthritis in nonhuman primates is very important, because it is known to share many similar immunological features with human disease due to the close phylogenetic relationship with humans. In addition, immunological treatments, using monoclonal antibodies to certain proteins shared by humans and monkeys, for example, can be performed in monkeys, with greater predictive value of the pathological roles and efficacy for humans of those proteins than in rodents. In the present study, we established CIA in cynomolgus monkeys, to whose IL-6R humanized anti-human IL-6R antibody, MRA, reacts, and then examined the anti-arthritic effects of MRA. MATERIALS AND METHODS

Monkeys Cynomolgus monkeys (Macaca fascicularis) from China, 3.2 to 5.4 years old, were housed individually under SPF conditions at 24 ⫾ 2°C and relative humidity of 55 ⫾ 5% (Table 1). The monkeys were given solid food PS R for specific pathogen-free monkeys (Oriental Yeast Co. Ltd.) at 100 g/hour/day, commercially available bananas at half/hour/day, and tap water ad libitum from an autosupply system. The bleeding and the treatment of monkeys were conducted according to “The standard of bleeding and maintenance of experimental animals” and “The standard of animal experiment procedure” established by the Experimental Animal Maintenance Committee of Chugai Pharmaceutical Co., which are in accordance with the guidelines of the Japanese Association for Laboratory Animal Science. Since there were many items based on macroscopic or subjective observation/measurement in the data to be collected in this study, the study was conducted as a blinded study with a controller to avoid the possible bias of examiners.

319 1521-6616/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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TABLE 1 Monkey List Treatment Vehicle

MRA (1 mg/kg)

MRA (10 mg/kg)

No.

Sex

Age (years)

1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

Female Female Male Female Female Female Male Female Female Female Female Male Female Female Female

4.6 5.4 4.3 4.2 5.4 4.3 4.2 3.2 5.3 5.4 4.1 4.1 5.2 3.6 5.2

the mark, to determine the volume of the hand or foot from the increase in volume. Measurement was carried out three times, and the median value was taken. The sum for the four limbs was normalized by body weight to obtain data for each animal. Inflammatory Parameters Blood samples were collected from the femoral vein every 2 weeks, starting from just before the first immunization, and the blood, plasma, and serum obtained were subjected to measurement of the erythrocyte sedimentation rate (ESR; Benoject ESR kit, Terumo, Tokyo), fibrinogen (assay performed by Mitsubishi Chemical BCL), and C-reactive protein (CRP) (TIA test-CRP Nissui, Nissui Pharmaceutical, Tokyo). Anti-CII Antibody

Experimental Schedule MRA was expressed in Chinese hamster ovary cells and purified on protein A. It was dissolved in phosphate buffer and administered intravenously once a week for 13 weeks. For the vehicle group, the same volume of phosphate buffer was administered in the same way. MRA recognizes the IL-6R of cynomolgus monkey and blocks the signal transduction of IL-6 (9, 10). Induction of Collagen-Induced Arthritis Bovine type II collagen (CII) was purchased from Cosmo Bio (Tokyo, Japan) and dissolved overnight at 4°C in 0.1 M acetic acid solution to make a concentration of 4 mg/mL. The collagen solution was emulsified in an equal volume of complete adjuvant H37 Ra (DIFCO Laboratories). Monkeys were immunized with 1 mL of the cold emulsion by 10 intradermal injections on the back and 1 injection into the base of the tail. Four weeks later, monkeys were boosted with CII by 20 intradermal injections on the back and 1 into the base of the tail. Clinical Assessment of Arthritis Rigidity of all joints of the fingers and toes (proximal, middle, distal), carpus (tarsus), elbow (knee), and palm (heel) were investigated and scored, with 1 point allocated for each joint which demonstrated symptoms, without consideration of severity. It was possible to score a total of 64 points. Swelling was analyzed by measuring the volume of the hands and feet by water immersion. The carpal or tarsal apophysis was marked with a marker and immersed into a cylinder containing water, to the depth of

Serum levels of IgG anti-CII antibody were measured by ELISA. Briefly, serum diluted in PBS containing 1% BSA (DB) was added to each well of the immunoplate (Nunc-Immunoplate, MaxiSorp, Roskilde, Denmark), which was precoated with 5 ␮g/mL of bovine CII, and incubated for 2 h at room temperature. After the wells were washed, bound IgG was measured using peroxidase-conjugated goat antibody to monkey IgG (Organon Teknika, West Chester, PA). Substrate (o-phenylenediamine) was added, and the colorimetric reaction was measured with a microplate reader Model 3550 (Bio-Rad) at 490 nm. The IgG concentration was calculated from a standard curve generated using a pooled vehicle-treated monkey serum defined as 1000 units/mL. Anti-MRA Antibody Serum levels of anti-MRA antibody were determined by ELISA. Briefly, serum was added to each well of the immunoplate (Nunc), which was precoated with 5 ␮g/mL of MRA, and incubated for 2 h at room temperature. After the wells were washed, biotin-conjugated humanized PM-1 was added to the wells for a further 2 h at room temperature. After washing, alkaline phosphatase-conjugated streptavidin (Pierce, Rockford, IL) was added to the wells and incubated for 2 h. After washing, substrate (Sigma 104) was added, and the colorimetric reaction was measured using a microplate reader (Bio-Rad) at 405 nm. As there was no anti-MRA antibody standard available, the content was expressed as absorbency. Serum MRA Levels Serum MRA levels were measured by ELISA. Briefly, the wells of an immunoplate precoated with

ANTI-IL-6R ANTIBODY SUPPRESSES CIA IN MONKEY

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FIG. 1. Clinical parameters. (A) Rigidity of joints was investigated and a score of 1 point was given for each joint which demonstrated symptoms, without consideration of severity. It was possible to gain a total score of 64. For the statistical analysis, the data were the posttreatment value subtracted from the pretreatment value. Statistical significance was analyzed by the nonparametric Dunnett test; P ⬍ 0.05, vehicle vs 10 mg/kg MRA. (B) Swelling was determined by measuring the volume of both hands and feet by water immersion. The sum for four limbs was normalized by the body weight, to calculate the data for each animal. For the statistical analysis, the data were the posttreatment values divided by the pretreatment values. Statistical significance was analyzed by the parametric Dunnett test; P ⬍ 0.05, vehicle vs 10 mg/kg MRA. Each symbol indicates individual data of five monkeys, except for 12 and 14 weeks of vehicle groups (four monkeys).

anti-human IL-6 receptor monoclonal antibody (MT18) (5 ␮g/mL) were washed and then blocked with DB at room temperature for 2 h. After incubation, DB was removed and then human soluble IL-6 receptor (SR344) (100 ng/mL) was added to wells and left at room temperature for 2 h. After washing, serum was added to wells and incubated for a further 2 h. After the wells were washed, bound anti-MRA antibody was measured using alkaline phosphatase-conjugated goat anti-human IgG (Biosource, Camarillo, CA). Substrate (Sigma 104) was added, and the colorimetric reaction was measured with a microplate reader (Bio-Rad) at 405 nm. The MRA concentration was calculated from an MRA standard curve. Radiographic Examination Radiographic projections were made of hands and feet. The first projections were made before collagen immunization, and from 4 weeks projections were

made at 2-week intervals. Radiographic grading of the four limbs was carried out by a blinded investigator, using a grading system of 0 –3: 0, no change; 1, the obscurity of the epiphysis progresses further and the articular cavity disappears. 2, the number of joints lacking articular cavities increases, the boundary between bones becomes unclear, and swelling of the bone causing closure of the epiphysis; obscurity of the surroundings also progresses; 3, these changes advance further, bones are twisted, and major distortion in configuration occurs. Histological Examination At the end of the examination (14 weeks), the four limbs of all animals were fixed, decalcified, embedded, sectioned, and stained with hematoxylin– eosin. Histological grading of four limbs was carried out by a blinded investigator, using a grading system of 0 –3: 0, no change; 1, minimal change in synovium, cartilage,

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FIG. 2. Inflammatory parameters. (A) ESR: For the statistical analysis, the data were the posttreatment values subtracted from the pretreatment values. Statistical significance was analyzed by the nonparametric Dunnett test; P ⬍ 0.05, vehicle vs 10 mg/kg MRA. (B) Serum CRP concentration: For statistical analysis, the data were the posttreatment values subtracted from the pretreatment values. Statistical significance was analyzed by the nonparametric Dunnett test; P ⬍ 0.05, vehicle vs 10 mg/kg MRA. (C) Plasma fibrinogen concentration: For statistical analysis, the data were the posttreatment values subtracted from the pretreatment values. Statistical significance was analyzed by the parametric Dunnett test; P ⬍ 0.05, vehicle vs 10 mg/kg MRA. Each symbol indicates individual data of five monkeys, except for 12 and 14 weeks of vehicle groups (four monkeys).

and bone; 2, moderate change in synovium, cartilage, and bone; 3, marked change in synovium, cartilage, and bone. Statistical Analysis For the statistical analysis, the data other than for swelling rates used were the posttreatment values subtracted from the pre-treatment values. For swelling rates, the data were the posttreatment values divided by the pretreatment values. For each dose level, the data were first analyzed by ANOVA to determine the interaction between dose level and time. As no such interaction was observed, the data were pooled from 2 to 14 weeks. Then the normal distribution was checked

by the Shapiro–Wilk test, after which the data with normal distribution were tested by the parametric Dunnett test, while the data without normal distribution were tested by the nonparametric Dunnett test, in both cases against the vehicle group. The anti-CII antibody level was analyzed by the parametric Dunnett test. The radiographic examination was analyzed by the ␹ 2 test. RESULTS

Clinical Observation All vehicle-treated monkeys showed clinical signs of arthritis 4 weeks after the first collagen immunization. The numbers of stiff joints and swelling reached

323

ANTI-IL-6R ANTIBODY SUPPRESSES CIA IN MONKEY

maxim at 8 and 6 weeks, respectively. Thereafter, arthritis decreased quickly. In contrast, 10 mg/kg of MRA inhibited the development of arthritis significantly. In this group, four of five monkeys had no signs of stiff joints (P ⬍ 0.05 vs controls) and swelling (P ⬍ 0.05 vs controls) throughout the experiment. In the 1 mg/kg group, two of five monkeys showed development of neither stiff joints nor swelling, although there were no significant differences (Fig. 1). One case in the vehicle group was subjected to euthanasia in extremis at 8 weeks.

TABLE 2 Radiographic Examination Weeks after first collagen immunization Treatment Vehicle

MRA (1 mg/kg)

Blood Parameters In the vehicle group, dramatic increases in CRP and fibrinogen were observed after 2 weeks, and a clear increase in ESR was observed after 4 weeks, following the first immunization. These effects were transient, as with the arthritis symptoms, and the situation returned to close to normal after 14 weeks. On the other hand, 10 mg/kg of MRA almost completely suppressed them (Fig. 2). Radiographic Study Aberration in X-ray photographs began to be observed after 4 weeks in the vehicle group, and after 6 weeks the epiphysis became obscure, due to the loss of cavitas articular in all cases. In contrast, one case administered 1 mg/kg and four cases administered 10 mg/kg showed no change throughout the study (Table 2). These findings were analyzed using the ␹ 2 test, which indicated that the 10 mg/kg MRA group was significantly different from the vehicle group (6 weeks, P ⬍ 0.05; 8 weeks, P ⬍ 0.05; 10 weeks, P ⬍ 0.05). Histological Study Histological study was carried out at the end of the examination (14 weeks). As at this point, the severity of arthritis was decreased. Although synovial proliferation and pannus formation, infiltration by neutrophils, angiogenesis, and cartilage and bone destruction were observed in all monkeys in the vehicle group, these changes were weak in some cases. Under this condition, however, histological changes were not observed in two monkeys administered 1 mg/kg of MRA and four monkeys administered 10 mg/kg of MRA (Table 3). Serum Anti-CII Antibody In the control group, serum IgG anti-bovine CII antibody appeared in one of five monkeys at 2 weeks and in all monkeys at 4 weeks and then increased, reaching the maximum at 8 weeks after collagen immunization.

MRA (10 mg/kg) b

No. 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

0 a

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

4

6

8

10

12

14

1 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1 1 1 1 1 0 0 1 1 0 0 0 0 1 0

2 2 1 1 1 1 0 1 1 0 0 0 0 1 0

2 2 1–2 1 1 2 1 2 2 0 0 0 0 1 0

2

2

2 1 1 2 1 3 3 0 0 0 0 2 0

2 1 1 2 1 3 3 0 0 0 0 3 0

a

Numeral indicates radiographic gradings. Radiographic projections were made of hands and feet. Radiographic grading of four limbs was determined by a blinded investigator using a grading system of 0 –3. b Statistical significance was analyzed by ␹ 2 test; P ⬍ 0.05 (6 weeks), P ⬍ 0.05 (8 weeks), P ⬍ 0.05 (10 weeks).

The appearance of anti-CII antibody in the 1 and 10 mg/kg MRA groups was delayed, with two and three monkeys in the 1 and 10 mg/kg groups, respectively, having no antibody even after 4 weeks. However, antibody levels reached the same level as that in the vehicle group at 8 weeks (Table 4). Serum Anti-MRA Antibody Anti-MRA antibody was detectable in 5/5 and 1/5 in the 1 and 10 mg/kg groups, respectively (Table 5). Serum MRA Levels Serum MRA concentrations in the 10 mg/kg group were maintained at high levels (over 100 ␮g/mL). In one monkey with the anti-MRA antibody it became undetectable at 6 weeks. However, in the case of 1 mg/kg groups, it was hardly detected (Table 6). DISCUSSION

Our results clearly show that the administration of 10 mg/kg MRA starting from the day of CII immunization suppresses the onset of clinical symptoms of arthritis, and increases in inflammatory parameters (CRP, fibrinogen, and ESR) are inhibited. To our knowledge, this is the first paper indicating that IL-6

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TABLE 3 Histological Examination Third finger R

Third toe L

R

L

Wrist

Ankle

Elbow

Knee

No.

PIP

DIP

PIP

DIP

PIP

DIP

PIP

DIP

R

L

R

L

R

L

R

L

1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

0 0 0 0 0 3 0 2 2 0 0 0 0 2 0

2 0 ND 0 1 2 0 2 2 0 0 0 0 2 0

0 0 1 0 0 2 0 2 2 0 0 0 0 1 0

0 2 2 0 0 2 0 2 3 0 0 0 0 0 0

0 1 2 1 0 1 0 2 1 0 0 0 0 3 0

2 1 1 0 0 2 0 1 2 0 0 0 0 3 0

2 1 2 0 0 2 0 3 3 0 0 0 0 3 0

2 2 2 0 0 2 0 2 2 0 0 0 0 1 0

2 1 1 0 0 1 0 1 2 0 0 0 0 2 0

1 2 2 0 1 1 0 2 2 0 0 0 0 1 0

2 2 3 2 0 1 0 1 2 0 0 0 0 2 0

3 1 2 1 0 1 0 2 2 0 0 0 0 2 0

1 1 0 0 1 0 0 1 2 0 0 0 0 0 0

1 1 0 0 2 0 0 1 2 0 0 0 0 0 0

2 1 0 0 0 0 0 2 2 0 0 0 0 2 0

2 0 0 0 0 0 0 2 2 0 0 0 0 2 0

Vehicle

MRA (1 mg/kg)

MRA (10 mg/kg)

Note. At the end of the experiment (14 weeks), all monkeys except one in the vehicle group were sacrificed and subjected to histological examination. The data were collected at 8 weeks. Histological grading of four limbs was determined by a blinded investigator using a grading system of 0 –3. R, right; L, left; PIP, proximal interphalangeal; DIP, distal interphalangeal; ND, not done.

might be an essential cytokine in the development of CIA in monkeys. The monkey CIA model is useful for the evaluation of certain immunomodulating therapies, such as those involving monoclonal antibodies to certain proteins shared by humans and monkeys (6 – 8). Although MRA is a humanized anti-human IL-6R, it was previously reported to have the ability to cross-react with monkey IL-6R and to inhibit the proliferation of T cells and antibody production by B cells from cynomolgus monkeys, which are induced by IL-6 (10). Furthermore, administration of MRA to cynomolgus monkeys completely inhibited IL-6-induced increases in platelet counts and CRP levels in peripheral blood (9). These results indicate that MRA binds to cynomolgus monkey IL-6R and blocks signal transduction of IL-6. ThereTABLE 4 Serum Levels of IgG Anti-Collagen Antibody Week

Vehicle

MRA (1 mg/kg)

MRA (10 mg/kg)

2 4 6 8 10 12 14

24.7 ⫾ 24.7 223.8 ⫾ 66.6 582.3 ⫾ 189.1 791.4 ⫾ 318.1 669.5 ⫾ 297.4 673.2 ⫾ 240.4 641.0 ⫾ 242.7

0.0 ⫾ 0.0 84.4 ⫾ 51.8 275.8 ⫾ 141.3 506.1 ⫾ 154.5 620.9 ⫾ 255.1 537.5 ⫾ 223.6 557.7 ⫾ 255.6

0.0 ⫾ 0.0 59.6 ⫾ 59.6 211.1 ⫾ 83.7 549.6 ⫾ 249.0 524.7 ⫾ 159.0 493.3 ⫾ 120.6 463.5 ⫾ 90.9

Note. IgG anti-collagen antibody levels were measured by ELISA. Each value indicates the mean and SE of monkeys. Statistical significance was analyzed by parametric Dunnett test.

fore, the suppression of monkey arthritis by MRA may be due to monkey IL-6R blockage. Anti-tumor necrosis factor-␣ (TNF␣) and anti-IL-1 therapies are effective in the treatment of CIA (11, 12) as well as RA patients (13, 14). Although IL-6 is thought to be located downstream of cytokine production, i.e., both TNF␣ and IL-1 induce IL-6 production, our present data clearly suggested that IL-6 is a main target for RA therapy besides TNF␣ and IL-1. In fact, anti-IL-6 antibody showed beneficial effect in the therapy of RA patients (15). Although MRA is a humanized antibody of mouse antibody, which reduces antigenicity for human use, it is still a heterogeneous protein in monkeys. Anti-MRA antibodies were detected in 5/5 and 1/5 monkeys in the groups treated with 1 and 10 mg/kg of MRA, respectively. Although the precise mechanism of this phenomenon is obscure, similar observations were also obtained with therapy using anti-TNF␣ antibody in humans (16). There are two ways to explain how a lower dose (1 mg/kg) of MRA more readily induce antibodies to the administered antigen than a higher dose (10 mg/kg). One possibility is that MRA itself at a higher dose might suppress anti-MRA antibody production, since IL-6 is known to play a role in the differentiation of B cells to antibody-producing cells (1). However, this is unlikely, because MRA did not show clear suppression of anti-CII antibody production in this experiment. Another possibility may be the induction of immune tolerance to MRA. It is known that intravenous administration of high dose protein can

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ANTI-IL-6R ANTIBODY SUPPRESSES CIA IN MONKEY

TABLE 5 Serum Anti-MRA Antibody Levels Weeks after first collagen immunization Treatment Vehicle

MRA (1 mg/kg)

MRA (10 mg/kg)

No.

0

2

4

6

8

10

12

14

0.122

0.133

1

0.128

0.141

0.133

0.139

0.126

0.134

2

0.114

0.120

0.116

0.114

0.113

0.117

3

0.105

0.107

0.094

0.102

0.092

0.101

0.102

0.105

4

0.112

0.119

0.106

0.113

0.104

0.111

0.109

0.119

5

0.108

0.127

0.103

0.120

0.100

0.121

0.103

0.122

1

0.112

0.480

0.383

1.077

1.765

2.128

1.930

2.063

2

0.099

0.113

0.105

0.155

0.110

0.274

0.397

0.499

3

0.116

0.805

0.472

0.355

0.537

0.869

0.914

0.862

4

0.110

2.818

2.843

0.380

0.294

0.414

1.261

2.090

5

0.109

0.324

0.140

0.157

0.129

0.148

0.141

0.278

1

0.103

0.107

0.097

0.102

0.095

0.102

0.102

0.104

2

0.110

0.140

0.108

0.132

0.106

0.131

0.107

0.134

3

0.111

0.136

0.105

0.128

0.105

0.124

0.104

0.127

4

0.111

0.120

0.104

0.142

0.147

0.177

0.273

0.586

5

0.109

0.120

0.105

0.114

0.102

0.111

0.104

0.113

Note. Serum levels of anti-MRA antibody were measured by ELISA. The data expressed the absorbance at 405 nm. Values over 0.150 were assessed as positive.

induce immune tolerance to administered protein. Similar observations were also obtained with therapy using anti-TNF␣ antibody in humans (16). It is noteworthy that anti-MRA antibodies may be produced less in humans, because MRA is a humanized antibody, that is an autologous protein. The efficacy of MRA in monkeys with anti-MRA antibodies was much weaker than in monkeys without anti-MRA antibodies. Furthermore, antibodies to MRA were readily produced in all monkeys administered 1 mg/kg of MRA, and MRA showed only a slight antiarthritic effect. These facts suggest that anti-MRA an-

tibodies suppress the anti-arthritic effect of MRA. This is supported by the fact that MRA in sera could not be measured, because of blocking by anti-MRA antibodies. In this study, the production of IgG anti-CII antibody was delayed for 2 weeks by the administration of MRA. However, the maximum level of anti-CII antibody was not decreased. Monkeys which were treated with 10 mg/kg of MRA, with no sign of arthritis, had as high levels of anti-CII antibody as vehicle-treated monkeys had. These data suggest that IL-6 only partially plays a roll in anti-CII antibody production in monkey. In

TABLE 6 Serum MRA Levels Weeks after first collagen immunization Treatment MRA (1 mg/kg)

MRA (10 mg/kg)

No.

2

4

1 2 3 4 5 1 2 3 4 5

— — — — — 108.1 83.7 79.1 102.2 115.8

— — — — — 250.0 93.0 122.2 12.4 119.3

6 — — 0.104 — — 148.6 73.2 149.3 — 170.7

Note. Serum MRA levels (␮g/mL) were measured by ELISA. (—) Not detectable.

8

10

12

14

— — — — — 172.7 72.1 151.6 — 137.1

— — — — — 113.6 38.9 156.0 — 89.6

— — — — — 169.9 43.1 198.5 — 101.2

— — — — — 127.5 40.1 112.6 — 109.6

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contrast, we reported that anti-mouse IL-6R antibody significantly suppressed IgG anti-CII antibody production in a mouse CIA model (5). The cause of this difference between mouse and monkey models is not clear. IL-6 is implicated in the development of T helper 1 (Th1) cells (17, 18). In fact, we found that IL-6 played an essential role in the induction of Th1 reaction, such as DTH reaction. As CIA is thought to be a Th1 type model (19, 20), MRA might inhibit CIA via the suppression of Th1 cell development. Further studies to verify this mechanism of action are being carried out. In conclusion, we report that IL-6 plays an important role in monkey CIA and that MRA may be an attractive agent for the treatment of RA. ACKNOWLEDGMENTS The authors thank Dr. Keiji Terao (National Institute of Infectious Disease), Dr. Michiko Tsuji (Jikei University School of Medicine), and Professor Katsuyuki Fujii (Jikei University School of Medicine) for thoughtful advice on establishing collagen arthritis in monkey. We also thank Mr. Toyokazu Matsu-ura, Mr. Hajime Sano, Ms. Akiko Hiura, and Ms. Mayumi Miyaguchi for assistance in conducting the experiments. REFERENCES 1. Akira, S., Taga, T., and Kishimoto, T., Interleukin-6 in biology and medicine. Adv. Immunol. 54, 1–78, 1993. 2. Hirano, T., Matsuda, T., Turner, M., Miyasaka, N., Buchan, G., Tang, B., Sato, K., Shimizu, M., Maini, R., Feldmann, M., and Kishimoto, T., Excessive production of interleukin 6/B cell stimulatory factor-2 in rheumatoid arthritis. Eur. J. Immunol. 18, 1797–1801, 1988. 3. Houssiau, F. A., Devogelaer, J-P., van Damme, J., de Deuxchaisnes, C. N., and van Snick, J., Interleukin-6 in synovial fluid and serum of patients with rheumatoid arthritis and other inflammatory arthritides. Arthritis Rheum. 31, 784 –788, 1988. 4. Madhok, R., Crilly, A., Watson, J., and Capell, H. A., Serum interleukin 6 levels in rheumatoid arthritis: Correlations with clinical and laboratory indices of disease activity. Ann. Rheum. Dis. 52, 232–234, 1993. 5. Takagi, N., Mihara, M., Moriya, Y., Nishimoto, N., Yoshizaki, K., Kishimmoto, T., Takeda, Y., and Ohsugi, Y., Interleukin-6 receptor blockage ameliorates joint disease in murine collagen-induced arthritis. Arthritis Rheum. 41, 2117–2121, 1998. 6. Yoo, T. J., Kim, S-Y., Stuart, J. M., Floyd, R. A., Olson, G. A., Cremer, M. A., and Kang, A. H., Induction of arthritis in monkeys by immunization with type II collagen. J. Exp. Med. 168, 777–782, 1988. 7. Cathcart, E. S., Hayes, K. C., Gonnerman, W. A., Lazzari, A. A., and Franzblau, C., Experimental arthritis in a nonhuman primate. Lab. Invest. 54, 26 –31, 1986. Received October 6, 2000; accepted November 15, 2000

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