Elsevier IMLET 01484

Involvement of interleukin-l-like factor(s) in type II collageninduced arthritis in mice T. Kasama l, K. Kobayashi 1, H. Kanemitsu l, K. Nakatani l, S. Kaga 1, N. Yamagata 1, M. Negishi l, H. Ide 1, T. Takahashi 1 and Y. Niwa 2 lFirst Department of Internal Medicine, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo, and 2Niwa Institute for Immunology, Kochi, Japan (Received 6 June 1990; accepted 3 July 1990)

1. Summary

To explore the role of interleukins in development of arthritis, we induced collagen-induced arthritis in mice and examined interleukin activities in the inflamed joints. Arthritis developed in 90070 of mice 4 - 5 weeks after primary immunization with type II collagen. Joint extracts from mice with collageninduced arthritis contained high levels of interleukin 1 (IL-1)-like activity but not interleukin 2 (IL-2) or interleukin 4 (IL-4) activity. IL-l-like activities in the lesions were correlated with development of arthritis assessed by joint swelling and erythema. These results suggest that IL-l-like factor(s) may participate in the etiopathogenesis of collagen-induced arthritis in mice.

Evidence is presented suggesting that IL-l-like factor(s) is involved in the development of collageninduced arthritis in mice. 3. Materials and Methods

3.1. Induction of collagen-induced arthritis

Collagen-induced arthritis is a chronic inflammatory arthropathy induced by immunizing rodents with type II collagen, the major matrix protein of hyaline cartilage [1]. The model displays several characteristics similar to human rheumatoid arthritis [1- 3]. The onset of collagen-induced arthritis is associated with the development of both cellular and humoral immune responses to type II collagen, although the role of cytokines is less clear [1-3].

Male DBA/1J mice were purchased from the Jackson Laboratory, Bar Harbor, ME. Mice were immunized at age of 8-14 weeks. Chick type II collagen was purchased from Genzyme (Boston, MA). Type II collagen-induced arthritis in mice was elicited by the method described previously [ 2 - 4]. Briefly, mice were immunized by intradermal injection of 100/zg of type II collagen emulsified in complete Freund's adjuvant (Difco Laboratories, Detroit, MI). Mice were boosted intraperitoneally with 100/zg of type II collagen dissolved in 0.1 M acetic acid. As controls, mice were injected with complete Freund's adjuvant and acetic acid. Mice were observed daily for the presence of distal joint swelling and erythema. Swelling was quantitated by measuring thickness of footpads with an engineer's micrometer (Mitsutoyo Co., Tokyo, Japan). The percent increase of swelling was calculated by measuring the difference in thickness before and after the onset of arthritis.

Keywords: Collagen-induced arthritis; Interleukin-l-likeactivity

3.2. Histologic evaluation

Correspondence to: Tsuyoshi Kasama, M.D., The First Department of Internal Medicine, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan.

Histologic sections were made from each decalcified joint tissue. These were prepared at a thickness

2. Introduction

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of 5/xm and were stained with hematoxylin and eosin.

tained approx. 1.0-2.5 mg protein/ml of PBS, as measured by the method of Lowry et al. [5].

3.3. Aqueous joint extracts

3.4. Assay for cytokines

Joint tissues were separated below the ankle joint by removing skin. Weights of joint tissues were 150-200 mg. Joints were homogenized in phosphate-buffered saline (PBS; 10% w/v) using a Polytron (Brinkmann Instruments, Westbury, NY) for 1 min. During these procedures, the tissue was kept on ice. The homogenized tissues were then centrifuged at 2000×g for 30 min. The supernatants were sterilized with a millipore filter (0.45/zm) and were stored at - 8 0 °C until use. The extracts con-

The thymocyte proliferation activity of IL-1 was determined by its capacity to stimulate C 3 H / H e J mouse thymocytes in the presence of phytohemagglutinin-P (PHA-P: Difco) as described [6]. Because C T L L cells are sensitive to both IL-2 and IL-4 [7, 8], these activities were assayed by their ability to stimulate proliferation of CTLL cells. The CTLL was provided by K. A. Smith. Proliferation was determined by incorporation of [3H]thymidine (pulsed with 18.5 kBq = 0.5 #Ci/well, specific ac-

Fig. 1. C o m p a r i s o n between an arthritic m o u s e hindpaw (A and B) with that of a control m o u s e (C and D). Grossly, the knee joint of a mouse with arthritis was swollen and injected (A). Histologically, pannus formation, infiltration of inflammatory cells and capillary vascularization of the joint were found (B). In contrast, minimal reactions were found in a control mouse (C and D). Arthritis developed on day 30; this photograph was taken 72 h later (H&E stain, x 100).



ti~,ity 248 GBq = 6.7 Ci/mmol, New England Nuclear, Boston, MA) for final 4-h incubation. In all assays, a half-maximal unit was reported [7]. Unit activities per mg protein of joint tissues were determined, and were expressed as the mean ___ SEM in triplicate. As positive controls in cytokine assays, we used recombinant human IL-I/~ (specific activity: 2 × 107 U/mg, Otsuka Pharmaceutical Co., Tokushima, Japan), recombinant human IL-2 (specific activity: 1 x 107 U/mg, Shionogi Pharmaceutical Co., Osaka, Japan) and recombinant murine IL-4 (specific activity: 1 × l0 s U/mg, Genzyme, Boston, MA).


cularization of the joint and infiltration of inflammatory cells including macrophages, polymorphonuclear leukocytes and lymphocytes (Fig. 1B) were found. In contrast, minimal reactions were found in control mice (Fig. 1C and D). 4.2. IL-l-like activity in inflamed joints As shown in Fig. 2, IL-l-like activity was found in joint extracts prepared from mice with arthritis but not from control mice. The activity reached peak intensity 4 - 5 weeks after the primary immunization and then gradually decreased thereafter. The activity correlated well with disease activity assessed by joint swelling and histology. As shown in Table 1, no CTLL proliferation activity was detected in samples that had thymocyte proliferation activity as described above. Our failure to detect both IL-2 and IL-4 could be due either to a relative inability of joint cells to produce these lymphokines or the presence of inhibitory factors that interfere with the expression of IL-2 and/or IL-4 activity. We therefore examined the ability of the extracts to neutralize or inhibit

4. Results

4. l. Induction of arthritis Arthritis developed in 90% of the mice 4 - 5 weeks after the primary immunization with type II collagen. As shown in Fig. 1A, the knee joint of the mouse with arthritis was swollen and injected. Histologically, pannus formation, capillary vas-

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Weeks after Immunization Fig, 2. KineticsofsweilingandlL-l-likeactivityofthejoints.Arthritisdevelopedin90% o f t h e m i c e 4 - 5 weeksafter the primary immunization with type II collagen ( a ). In contrast, no joint swelling was found in control mice ( • ). IL-l-like activity was found in the joint extracts of arthritic mice ( o ), but not in those o f control mice ( • ). Data represent the m e a n +_ SEM in five separate experiments of 2 - 3 mice per each condition.


TABLE 1 Lack of CTLL proliferation activity in joint tissue extracts. Samples from

Control mice Mice with arthritis

Weeks after the immunization 1 - 4 weeks

5 - 8 weeks

Involvement of interleukin-1-like factor(s) in type II collagen-induced arthritis in mice.

To explore the role of interleukins in development of arthritis, we induced collagen-induced arthritis in mice and examined interleukin activities in ...
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