Human microvascular endothelial cell activation by IL-1 and TNF-alpha stimulates the adhesion and transendothelial migration of circulating human CD14+ monocytes that develop with RANKL into functional osteoclasts.

UNLABELLED: Circulating pre-OCs may be recruited to locally inflamed sites through specific interactions with activated microvasculature. We found that HMVECs stimulated the adhesion and TEM of circulating pre-OCs, in an ICAM-1- and CD44-dependent manner, leading to greater RANKL-induced OC formation and bone pit resorption. INTRODUCTION: Inflammation is critical for healing processes but causes severe tissue destruction when chronic. Local osteoclast (OC) formation and bone resorption may increase at inflammatory sites through multiple mechanisms, including direct stimulation by inflamed microvasculature of circulating OC precursor (pre-OC) migration through a blood vessel barrier into bone or joint tissue. How this might occur is not yet well understood. MATERIALS AND METHODS: Cytokine-activated human microvascular endothelial cell (HMVEC) monolayers, with or without IL-1 and TNF-alpha preactivation (24 h), were incubated in adhesion (1-3 h) or porous transwell transendothelial migration (TEM; 3 h) assays with human peripheral blood mononuclear cells (hPBMCs) or CD14+ monocyte or CD14- lymphocyte subsets. The number of cells that adhered or transmigrated, and their ability to thereafter develop with macrophage-colony stimulating factor (M-CSF) + RANKL into bone pit-resorbing OCs, were analyzed. Immunostaining and neutralizing antibodies to key cell adhesion molecules were used to determine their potential involvement in stimulated CD14+ monocyte TEM. RESULTS: M-CSF + RANKL caused OC and bone pit formation only from hPBMCs and CD14+ cells but not CD14- cells. Adhesion of hPBMCs or CD14+ cells but not CD14- cells was stimulated by cytokine preactivation of HMVECs and led to the full capture of all circulating pre-OCs capable of developing into OCs. Cytokine-preactivated HMVECs also promoted the postadhesion TEM of hPBMCs and CD14+ populations, resulting in markedly greater OC formation and bone pit resorption by transmigrated cells. Immunodetectable vascular cell adhesion molecule (VCAM-1), intercellular adhesion molecule (ICAM-1), and CD44 levels increased on cytokine-treated HMVEC surfaces, and neutralizing antibodies to ICAM-1 or CD44, but not VCAM-1 or platelet endothelial cell adhesion molecule (PECAM-1), inhibited stimulated CD14+ cell TEM through activated HMVECs. CONCLUSIONS: This is the first demonstration that cytokine-activated HMVECs efficiently capture and promote the TEM of circulating pre-OCs capable of differentiating into bone-resorbing OCs. Thus, direct pre-OC recruitment by activated microvasculature at inflammatory sites may significantly contribute to normal OC bone remodeling during fracture healing or exacerbate pathological bone loss in various chronic inflammatory disorders.

Stromal cell-derived factor-1 binding to its chemokine receptor CXCR4 on precursor cells promotes the chemotactic recruitment, development and survival of human osteoclasts.

Osteoclasts (Oc) derive from hematopoietic precursors present in the circulation and bone marrow, and they differentiate into multinucleated bone-resorbing cells in response to the dual essential signals receptor activator of NF-kappaB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) primarily provided by bone marrow stromal cells (BMSC) and osteoblasts (Ob). However, little is known about signals that direct Oc precursors from the circulation into bone or control their migration within the marrow. Stromal cell-derived factor-1 (SDF-1 or CXCL12) is a chemokine highly expressed by bone endothelium, BMSC, and immature Ob that is essential for the normal homing, early development, and survival of various hematopoietic progenitor cells. We investigated whether SDF-1 and its unique chemokine receptor CXCR4 were involved in regulating human Oc precursor chemotaxis, development, function, or survival. CXCR4 was highly expressed by freshly isolated human monocyte (MN) populations, in vitro generated Oc and Oc-like cells, and mature Oc isolated from human femoral bones. SDF-1 markedly stimulated the chemotactic recruitment of circulating human MN capable of generating bone-resorptive Oc, leading to a 4-fold increase in Oc formation and greater bone pit resorption after their M-CSF + RANKL induced differentiation compared to spontaneously migrating cells. SDF-1 also directly promoted early (but not later) stages of Oc development via stimulating precursor cell numbers, multinucleated cell fusion, increased cell size, and tartrate-resistant acid phosphatase (TRAP) activity in a similar, but non-additive, fashion to M-CSF + RANKL. While SDF-1 did not cause full development of bone-resorbing Oc or stimulate the resorptive function of mature Oc directly, it also did not interfere with any actions promoted by M-CSF + RANKL. In mature human Oc, SDF-1 proved equally as effective as M-CSF + RANKL for preventing Oc apoptosis induced by cytokine withdrawal. In both cases, Oc survival was accompanied by analogous rises in the mRNA ratios for anti-apoptotic Bcl-xL and Bfl-1 relative to pro-apoptotic Bax, and by marked protein suppression of the critical pro-apoptotic signal Bim. These findings demonstrate for the first time that SDF-1 chemoattracts circulating human Oc precursors capable of developing into bone-resorptive Oc, and that it can stimulate MN cell fusion and TRAP activity, mimic M-CSF + RANKL in early osteoclastogenic effects, substitute for M-CSF + RANKL in maintaining the survival of mature human Oc, and suppress Oc expression of Bim protein. Thus, high levels of SDF-1 produced by bone endothelium, BMSC, and Ob may selectively target circulating Oc precursors into bone and stimulate their marrow migration into suitable perivascular stromal sites for their early development, RANKL differentiation, and survival. Consequently, SDF-1 may be a key factor linking bone vascular cells, BMSC, Ob, and Oc in the normal homeostatic regulation of bone development and remodeling.