Methods: J774.1 cells were pretreated with or without alendronate for 24 h and washed twice with serum-free RPMI-1640 in 96-well flat-bottomed plates. Cells were incubated in the presence or absence of Pam3CSK4 (a Toll-like receptor (TLR) 2 agonist, 10 ng/ml) or Lipid A (a TLR4 agonist, 100 ng/ml) in RPMI-1640 containing 10% FBS for 24 h. The culture supernatants were analyzed by ELISA for secreted mouse monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α) and transforming growth factor-β1 (TGF-β1), and results were determined using a standard curve prepared for each assay. NF-κB activation was determined by the TransAM™ ELISA kit. Total Smad3 and Phospho-Smad3 were detected by flow cytometry. Data were analyzed using one-way analysis of variance and either the Bonferroni or Dunn method. Results are presented as the mean ± SE of triplicate wells.
Results: Pretreatment of J774.1 cells with alendronate decreased the production of TLR ligand-induced MCP-1 and MIP-1α but did not influence NF-κB activation. Thus, the alendronate-mediated decrease in chemokine production was independent of NF-κB activation. Although TGF-β1 is known to inhibit chemokine production by various cell types via Smad3 activation, pretreatment with alendronate did not increase TGF-β1 production by J774.1 cells incubated in the presence or absence of TLR ligands. However, alendronate directly activated Smad3.
Conclusion: Alendronate down-regulates MCP-1 and MIP-1α production in response to TLR2 and TLR4 agonist via Smad3 activation.