CCR1 Chemokines Promote the Chemotactic Recruitment, RANKL Development, and Motility of Osteoclasts and Are Induced by Inflammatory Cytokines in Osteoblasts

Abstract

Chemoattractants that recruit OC precursors to locally inflamed sites of resorption are not well known. A chemokine receptor, CCR1, was expressed in OC precursors and elevated in mature OCs, and its ligands promoted OC precursor recruitment, RANKL development, and OC motility. Cytokines induced OB release of such chemokines, which may therefore significantly contribute to inflammatory bone loss. Chemokines, primarily of two major (CXC, CC) families, are essential signals for the trafficking and localization of circulating hematopoietic cells into tissues. However, little is known about their potential roles in osteoclast (OC) recruitment, development, or function. Previously, we analyzed CXC receptors in murine OC precursors and found high expression of CXCR4 that mediated their stromal-derived factor-1(SDF-1)-induced chemotaxis and collagen invasion. Here, we investigated if CC receptors and ligands, which are elevated in inflammatory and other osteolytic diseases, also play important roles in the recruitment, formation, or activity of murine bone-resorptive OCs. CC chemokine receptor (CCR) mRNA expression was analyzed during OC formation induced by RANKL in murine RAW 264.7 cells and primary marrow cells. Corresponding CC chemokines were tested for their ability to elicit precursor chemotaxis or OC development, or to influence motility, bone resorption, adhesion, or survival in RANKL-differentiated OCs. Constitutive and inflammatory cytokine-induced release of the chemokines macrophage inflammatory protein-1alpha (MIP-1alpha) and regulated on activation, normal T-cell expressed and secreted (RANTES) was measured by ELISA for OCs, osteoblasts (OBs), and their precursor cells. CCR1 was expressed in murine marrow cells, the most prominent CCR in RAW cells, and upregulated by RANKL in marrow or RAW cells. Chemokines that bind CCR1 (MIP-1alpha, RANTES, and monocyte chemoattractant protein-3 [MCP-3]) were produced to varying degrees by murine OCs, OBs, and their precursors, and markedly increased by interleukin (IL)-1alpha and TNFalpha in differentiating OBs. RANTES, and especially MIP-1alpha, increased mature OC motility, but did not alter OC resorption activity, adhesion, or survival. All three chemokines stimulated chemotaxis of marrow or RAW cell precursors, leading to the greater formation of OCs (in number and size) after RANKL development of such chemoattracted marrow cells. All three chemokines also directly and dramatically enhanced OC formation in marrow cultures, through a pathway dependent on the presence of RANKL but without altering RANK expression. Pathological increases in secretion of these chemokines from activated OBs or other cells may potently stimulate the chemotactic recruitment and RANKL formation of bone-resorptive OCs, thereby exacerbating local osteolysis in multiple skeletal diseases.