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Figure 4


Fig. 4. Requirement of TLR4 for HMM-HA inhibition of osteoclast differentiation. (A) Blockade of HA effects by a TLR4-neutralizing Ab. Human PBMCs were cultured for 9 days with RANKL (200 ng/ml), M-CSF (30 ng/ml), HA (1 µg/ml) and a TLR4 Ab (1-10 µg/ml) or an isotype-matched control Ab (10 µg/ml). Cells were stained for TRAP, and TRAP+ MNCs were counted. **P<0.01 compared with HA-treated control. (B) Lack of HA effects in TLR4-mutant cells. BMMs from TLR4-mutant mice were cultured with M-CSF (30 ng/ml) and RANKL (100 ng/ml) together with HA (0.1-5 µg/ml) for 6 days. TRAP+ MNCs were counted. (C) Binding of HA to TLR4. HEK-293 cells were transfected with TLR2, TLR4, TLR5, TLR9, or a control (mock) plasmid. At 24 hours after transfection, cells were incubated with anti-CD44 (1 µg/ml, 1 hour) to block potential HA binding to CD44. Cells were then incubated with FITC-HA for 30 minutes and analyzed in a flow cytometer. Red line, TLR-transfected cells; black line, mock-transfected cells.





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