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Bone resorbing activity released from zymosan-activated mouse peritoneal macrophages--the role of prostanoids and
A Ohlin1, U Sjögren, U H Lerner
1Department of Orthopaedics, Malmö University Hospital, Lund University, Sweden.
Objective:
To study the effect of zymosan on the release of osteoclast stimulating activity from macrophages.
Materials:
Calvarial bones from neonatal mice and peritoneal macrophages were incubated in the absence and presence of zymosan for 72 h and supernatants harvested for subsequent analysis of bone resorbing activities and prostaglandin concentration.
Methods:
Bone resorption was assessed in vitro by analysing the release of 45Ca and 3H from neonatal mouse calvarial bones prelabelled in vivo by injections of [45Ca]CaCl2 or [3H]-proline. Prostaglandin E2 (PGE2) and I2 (PGI2) were analyzed using radioimmunoassays.
Results:
Supernatants from macrophages treated with zymosan stimulated the release of 45Ca and 3H. The amount of bone resorbing activity present in the macrophage supernatants was dependent on the concentration of zymosan (0.1-100 microg/ml), as well as the number of macrophages present. The 45Ca release induced by zymosan treated macrophages was inhibited by three different 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 filter with a cut off of 3000 Daltons. Time-course studies of the production of bone resorbing activity in macrophages showed that activity increased during the first hour of exposure to zymosan and then reached a plateau for 96 h. PGE2 and PGI2 release from macrophages was increased during the first three hours of exposure to zymosan. This prostanoid production, together with bone resorbing activity, was abolished by indomethacin. The bone resorbing activity present 3-72 h after zymosan exposure, however, was not inhibited by indomethacin. Bone resorption stimulated by conditioned media from zymosan treated macrophages after 3 h was inhibited by 60-75% in the presence of anti IL-1alpha, 0-20% by anti IL-1beta, and completely by antisera neutralizing both IL-1alpha and IL-1beta. In addition, an IL-1 receptor antagonist abolished the stimulatory effect of conditioned media from zymosan treated macrophages.
Conclusions:
These data indicate that treatment of mouse peritoneal macrophages with zymosan results in production of activities capable of stimulating bone resorption in vitro. The activity released initially appears to be due to a zymosan induced burst of prostanoid production, while the activity released during prolonged exposure to zymosan is due primarily to IL-1alpha.
Insights
Zymosan stimulates macrophages to release bone-resorbing factors. Initially, this involves prostanoids, followed by Interleukin-1alpha (IL-1alpha) with prolonged exposure, impacting osteoclast activity.
Area of Science:
- Immunology
- Cell Biology
- Bone Biology
Background:
- Macrophages play a role in bone remodeling.
- Zymosan is a known activator of immune cells.
Purpose of the Study:
- To investigate the effect of zymosan on macrophage-induced osteoclast stimulating activity.
- To characterize the factors released by zymosan-treated macrophages that affect bone resorption.
Main Methods:
- Neonatal mouse calvarial bones and peritoneal macrophages were cultured with zymosan.
- Bone resorption was measured by 45Ca and 3H release.
- Prostaglandin E2 (PGE2) and I2 (PGI2) levels were quantified.
- Interleukin-1 (IL-1) activity was assessed using specific antibodies and receptor antagonists.
Main Results:
- Zymosan-treated macrophages released bone-resorbing activity, dependent on zymosan concentration and macrophage number.
- Early bone resorption was linked to prostanoid production (PGE2, PGI2), inhibited by indomethacin.
- Later bone resorption was primarily mediated by IL-1alpha, as shown by inhibition with anti-IL-1alpha and IL-1 receptor antagonist.
Conclusions:
- Zymosan induces macrophages to produce factors that stimulate bone resorption in vitro.
- The initial bone resorption is mediated by prostanoids, while sustained resorption is driven by IL-1alpha.
- These findings highlight the complex interplay between immune activation and bone metabolism.