MATERIALS AND METHODS

Mice. BALB/c female mice (Seiwa Breeding Co. for Experimental Animals, Fukuoka, Japan), 6-8 weeks old, were used throughout this study. Synthetic MDP and its related compounds. MDP and 6-O-stearoyl-and 6-0-(2-tetradecylhexadecanoyl)-MDP(L18-MDP and B30-MDP, respectively) were gifts from Dr. A. Inoue(Research Laboratories, Daiichi Seiyaku Co., Tokyo). The synthesis of these compounds was described elsewhere(9, 10). Stereoisomers of MDP which were synthesized as described previously(9) were kindly supplied by Drs. S. Kusumoto and T. Shiba(Faculty of Science, Osaka University, Osaka, Japan). Induction of arthritis. Most synthetic compounds were dissolved in saline except for B30-MDP. B30-MDP which gave a fine suspension in physiological saline and 0.15 M phosphate-buffered saline, pH 7.2 (PBS), was dissolved in either 5% Nikkol HCO-60(Nikko Chemicals Co., Ltd., Tokyo) or 0.15 M phosphate buffer, pH 7.2 (PB). Animals in groups of five were intravenously injected into the tail with 4-100ƒÊg of B30-MDP in 0.2 ml of PB, PBS, or 5% Nikkol HCO-60 and of other test compounds in 0.1 ml of saline. Control animals were intravenously injected with either 0.1 ml of saline or with 0.2 ml of PB, PBS, and 5% Nikkol HCO-60, respectively.

Clinical evaluation. Mice were examined daily and graded for severity of arthritis by the method of Wood et al (16), which was developed for scoring the grade of adjuvant arthritis. Lesions of the extremities distal to the elbow or knee were graded on a scale of 0-4 based on the number of joints involved and the degree of erythema and swelling. The maximum score was thus 16. Histological evaluation. Histological examinations were performed on randomly chosen animals 20 hr or later after MDP and L18-MDP administration. Tissue specimens were fixed in 10% formalin, decalcified in formic acid-sodium citrate, embedded in paraffin by the conventional procedure, and stained with hematoxylin and eosin.