The Roles of CCL19 and CCL21 Chemokines in Osteoclast Migration and Bone Resorption

Abstract

OBJECTIVE: Bone resorption is a severe problem in inflammatory diseases such as periodontitis and rheumatoid arthritis (RA). Osteoclasts are responsible for bone resorption and CCL19 and CCL21 act as chemokines for several cell types. The purpose of this study was to investigate the role of CCL19 and CCL21 in bone resorption by osteoclasts.

METHODS: The levels of CCL19, CCL21 and CCR7 in RA and osteoarthritis patient samples were measured using ELISA. Data deposited to gene expression omnibus was also analyzed. The expression levels of these molecules and differentiation markers of osteoclasts were measured by real time polymerase chain reaction (PCR), western blotting or flow cytometry. Osteoclast differentiation was evaluated by tartate-resistant acid phosphatase staining and osteoclast migration was assessed with transwell assay and Oris migration assay kits. Resorption activity was performed with calcium phosphate-coated dishes or dentin slices, which were analyzed by von Kossa staining or confocal microscopy, respectively. Involvement of CCR7 was demonstrated with the small interference RNA system and contribution of GTPase Rho was investigated by the Rho pull-down assay. Collagen transplantation model was used to examine the in vivo effects of these chemokines.

RESULTS: The expression levels of CCL19, CCL21 and CCR7 were higher in RA patient samples compared to OA or normal samples. Bone marrow macrophages and osteoclasts expressed more CCR7 in response to the stimulation of TNF, IL-1 and LPS. CCL19 and CCL21 promoted the migration and resorption activity of both bone marrow macrophages and osteoclasts. Knock-down of CCR7 significantly reduced the CCL19- and CCL21-induced migration. Moreover, CCL19 and CCL21 stimulated small GTPase Rho and its downstream molecule, ROCK. Rho inhibitors suppressed both the migration and resorption activity of BMMs and osteoclasts. It demonstrates that the increase of migration and bone resorption by CCL19 and CCL21 was mediated by the CCR7/Rho axis. Collagen transplantation study showed that CCL19 and CCL21 can promote bone resorption in vivo.

CONCLUSION: This study indicates that under inflammatory conditions, osteoclast precursors increase the CCR7 expression. CCL19 and CCL21 are up-regulated in RA patients and enhance the osteoclast migration and resorption activity, resulting in severe bone destruction. This study suggests that neutralizing antibody for CCL19 and CCL21 or CCR7 knock-down are potential therapeutic strategies for periodontitis and RA.