Objectives: TNF-á is an important stimulator of bone resorption in inflammatory conditions through the induction of RANKL and the enhancement of RANKL signaling. The goals of this project were to determine if TNF-á can stimulate osteoclastogenesis in RAW 264.7 cells in the absence of RANKL and if TNF-á has some of its effects via the induction of prostaglandins (PGs) produced by cyclooxygenase (COX-2).

Methods: RAW 264.7 cells, a murine macrophage cell line that does not express RANKL, were plated at 1.2x10⁴ cells/well and treated with vehicle (control), RANKL (30 ng/ml), TNF-á (10 ng/ml) and RANKL+TNF-á. Some cultures were also treated with the nonsteroidal anti-inflammatory drugs (NSAIDs), indomethacin (1 µM) or NS-398 (0.1 µM), to inhibit PG production. Osteoclast-like cells (OCLs), defined as multinucleated (>3 nuclei) cells staining for tartrate resistant acid phosphatase (TRAP), were counted in 2-3 wells per treatment group on days 3-5 of culture. Medium PGE₂ was measured by EIA. COX-2 gene expression was analyzed by real-time PCR.

Results: No OCLs formed in control cultures. In 5/5 experiments TNF-á alone stimulated OCL formation, the mean number/well ranging from 8-190 on day 4 of culture. TNF-á appeared to increase mononuclear cell number as well. RANKL alone stimulated the formation of 906 ± 18 OCLs/well on day 4. Addition of TNF-á to RANKL increased RANKL-induced OCL number by 1.6-fold. Medium PG levels and COX-2 mRNA levels on days 3-4 of culture were increased by TNF-á alone compared to controls. Levels were not increased by addition of TNF-á to RANKL-stimulated cultures. Treatment with NSAIDs did not decrease OCL number in any treatment group.

Conclusions: TNF-á increased formation of OCLs independent of RANKL and enhanced RANKL-stimulated formation of OCLs in RAW 264.7 cells. These effects of TNF-á were independent of prostaglandins.

Back to the Mineralized Tissue Program
Back to the IADR/AADR/CADR 85th General Session and Exhibition (March 21-24, 2007)

Top Level Search