Bone resorbing activity released from zymosan-activated mouse peritoneal macrophages - the role of prostanoids and interleukin-1

Objective: To study the effect of zymosan on the release of osteoclast stim ulating activity from macrophages. Materials. Calvarial bones from neonatal mice and peritoneal macrophages we re incubated in the absence and presence of zymosan for 72 h and supernatan ts harvested for subsequent analysis of bone resorbing activities and prost aglandin concentration. Methods. Bone resorption was assessed in vitro by analysing the release of Ca-45 and H-3 from neonatal mouse calvarial bones prelabelled in vivo by in jections of [Ca-45]CaCl2 or [H-3]-proline. Prostaglandin E-2 (PGE(2)) and I -2 (PGI(2)) were analyzed using radioimmunoassays. Results: Supernatants from macrophages treated with zymosan stimulated the release of Ca-45 and H-3. The amount of bone resorbing activity present in the macrophage supernatants was dependent on the concentration of zymosan ( 0.1-100 mu g/ml), as well as the number of macrophages present. The Ca-45 r elease induced by zymosan treated macrophages was inhibited by three differ ent inhibitors of osteoclastic bone resorption (calcitonin, acetazolamide, amino bisphosphonate). The bone resorbing activity released by the zymosan- activated macrophages was lost after ultrafiltration using a filter with a molecular weight cut off of 30,000 Daltons, but retained when using a filte r with a cut off of 3000 Daltons. Time-course studies of the production of bone resorbing activity in macrophages showed that activity increased durin g the first hour of exposure to zymosan and then reached a plateau for 96 h . PGE(2) and PGI(2) release from macrophages was increased during the first three hours of exposure to zymosan. This prostanoid production, together w ith bone resorbing activity, was abolished by indomethacin. The bone resorb ing activity present 3-72 h after zymosan exposure, however, was not inhibi ted by indomethacin. Bone resorption stimulated by conditioned media from z ymosan treated macrophages after 3 h was inhibited by 60-75% in the presenc e of anti IL-1 alpha, 0 - 20 % by anti IL-1 beta, and completely by antiser a neutralizing both IL-1 alpha and IL-1 beta. In addition, an IL-1 receptor antagonist abolished the stimulatory effect of conditioned media from zymo san treated macrophages. Conclusions: These data indicate that treatment of mouse peritoneal macroph ages with zymosan results in production of activities capable of stimulatin g bone resorption in vitro. The activity released initially appears to be d ue to a zymosan induced burst of prostanoid production, while the activity released during prolonged exposure to zymosan is due primarily to IL-1 alph a.