CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY Vol. 83, No. 2, May, pp. 173– 178, 1997 Article No. II974333 RAPID COMMUNICATION The Effect of Dexamethasone on the Expression of Activated NF- kB in Adjuvant Arthritis PETER W. TSAO,TATSUO SUZUKI,RYUICHI TOTSUKA,TAKASHI MURATA,TOSHIKI TAKAGI, YASUSHI OHMACHI,* HISAKO FUJIMURA,* AND ISAO TAKATA Department of Inflammatory Diseases and *Department of Pathology and Toxicology, Lead Optimization Research Laboratory, Tanabe Seiyaku Co. Ltd., 2-2-50 Kawagishi, Toda-Shi, Saitama, Japan 335 NF-kB may be responsible for the potent anti-inflam- The transcription factor NF-kB plays a significant matory effects of steroids (3). role in inflammatory diseases. In this study we have Recently, a monoclonal antibody to the p65 subunit investigated the expression of activated NF-kB p65 of NF-kB was reported (4). This antibody detects only subunit in the rat adjuvant arthritis model in a 28-day the activated form of NF-kB because it binds to a region time-course experiment using immunohistochemistry. on the NF-kB molecule that is unmasked only after The expression of p65 was detected in the synovial lin- uncoupling of Ik-B. Using this antibody, Marok et al. ing layer and around the blood vessels in the inflamed reported the expression of activated NF-kB p65 subunit synovium as early as Day 3 post-adjuvant injection. in synovial tissues from arthritic patients (5). While The cells that expressed p65 in the synovial lining were the patient studies provide us with information in the thought to be macrophage-like synoviocytes. The ex- clinical situation, details about the expression of acti- pression was stronger in the injected hindpaw than vated NF-kB especially during the early phase of devel- that in the noninjected hindpaw. Dexamethasone oping arthritis are not available. Moreover, patients treatment at 1 mg/kg po (Days 0– 20) suppressed both will generally receive anti-inflammatory therapy which the hindpaw edema and increase in p65 expression. could influence the expression of activated NF-kB. Withdrawal of the treatment caused increases in both Therefore, the purpose of this study is twofold: first, p65 expression and paw volume. Together these sug- to investigate the expression of NF-kB in various stages gest that activated NF-kB was specifically expressed in the arthritic synovium and may play a significant of developing arthritis using the rat adjuvant arthritis role in the development of arthritis.  1997 Academic Press model in a time-course experiment; and second, to ex- amine the effect of dexamethasone, a NF-kB inhibitor, on the expression of NF-kB in this animal model. We INTRODUCTION report here the expression of activated NF-kB and the effect of dexamethasone in a rat adjuvant arthritis model using immunohistochemistry. The nuclear factor-k B (NF-kB) family of transcrip- tion factors plays a very important role in the inducible regulation of a variety of genes involved in inflamma- METHODS tory process. Various proinflammatory cytokines and mediators such as TNF-a, IL-1, and IL-6, and cellular Experimental Protocol adhesion molecules are shown to be regulated by NF- kB (1). NF-kB contains two subunits, p50 and p65. In Female Lewis rats, 8 weeks of age (160 { 10 g), were purchased from Charles River, Japan. After acclimati- its inactive form, NF-kB is located in the cytosol, cou- pled to the inhibitory molecule, Ik-B. Activation sig- zation the rats were randomly assigned into 15 groups (n Å 3 per group) according to treatment received (con- nals, such as those produced by cytokines or oxidative stress, cause Ik-B to dissociate from the NF-kB complex trol, adjuvant, and dexamethasone group) and time of sacrifice (Days 3, 7, 14, 21, and 28 post-adjuvant injec- (1). This allows the complex to translocate from the cytosol to the nucleus and binds to specific DNA se- tion). These time points were chosen based on our pre- vious experience with this model. The adjuvant group quence to initiate transcription. NF-kB activation can be blocked by a number of nonspecific inhibitors such received injection of 100 mg of heat-inactivated Myco- bacterium butyricum (Difco, U.S.A.) in 100 ml liquid as antioxidants, salicylates, and steroids (2). In particu- lar, steroids are among the most potent inhibitors paraffin in the right hindpaw. The dexamethasone group received same amount of adjuvant in the right known to inhibit NF-kB activity. This action on the 173 0090-1229/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved. AID Clin 4333 / a50e$$$201 04-03-97 22:54:43 clinal AP: Clin