Fig. (3) RANKL and TNF synergistically stimulate TNF mRNA expression by osteoclast precursor cells and decreased RANKL and TNF levels in joints of TNF-Tg/RANKL^-/- mice.(A) Spleen cells from WT mice were cultured with M-CSF (10 ng/ml) for 3 days to generate osteoclast precursors and then were treated with various amount of TNF, RANKL or in combination for 1 hour. (B) Spleen cells from WT and RANK^-/- mice were cultured with M-CSF and treated with TNF+RANKL (2.5 ng/ml) for 1 hour. Murine TNF mRNA expression was examined by real time RT-PCR. Values are presented as the mean plus SEM of 3 independent experiments done in triplicate, where the PBS control is arbitrarily set at 1. Similar results were obtained from an additional two pairs of WT and RANK^-/- mice. (C) Four-month-old WT, TNF-Tg, RANKL^-/-, and TNF-Tg/RANKL^-/- mice from the same litter were sacrificed and total RNA was extracted from wrist joints. Expression levels of murine TNF and RANKL mRNA were measured by real-time RT-PCR. *p< 0.05 vs WT cells or mice and #p< 0.05 vs TNF-Tg mice.