The receptor activator of NF-B (RANKL) is the essential signal required for full osteoclast (OC) development, activation, and survival. RANKL is highly expressed in areas of trabecular bone remodeling and inflammatory bone loss, is increased on marrow stromal cells or osteoblasts by osteotropic hormones or cytokines, and is neutralized by osteoprotegerin (OPG), a soluble decoy receptor also crucial for preventing arterial calcification. Vascular endothelial cells (VEC) are critically involved in bone development and remodeling and influence OC recruitment, formation, and activity. Although OCs develop and function in close association with bone VEC and sinusoids, signals mediating their interactions are not well known. Here, we show for the first time that human microvascular endothelial cells (HMVEC) express transcripts for both RANKL and OPG; inflammatory cytokines tumor necrosis factor-␣ and interleukin-1␣ elevate RANKL and OPG expression 5-40-fold in HMVEC (with an early OPG peak that declines as RANKL rises), and RANKL protein increases on the surface of tumor necrosis factor-␣-activated HMVEC. Cytokine-activated HMVEC promoted the formation, fusion, and bone resorption of OCs formed in co-cultures with circulating human monocytic precursors via a RANKLmediated mechanism fully antagonized by exogenous OPG. Furthermore, paraffin sections of human osteoporotic fractured bone exhibited increased RANKL immunostaining in vivo on VEC located near resorbing OCs in regions undergoing active bone turnover. Therefore, cytokine-activated VEC may contribute to inflammatorymediated bone loss via regulated production of RANKL and OPG. VEC-derived OPG may also serve as an autocrine signal to inhibit blood vessel calcification.The receptor activator of NF-B ligand (RANKL), 1 also known as osteoprotegerin ligand (OPGL), osteoclast differentiation factor, or TNF-related activation-induced cytokine (TRANCE), is a recently discovered transmembrane molecule of the tumor necrosis factor (TNF) ligand superfamily that is highly expressed in lymphoid tissues and trabecular bone, particularly in areas associated with active bone remodeling or inflammatory osteolysis (1-4). RANKL is the essential and final common signal required both in vitro and in vivo for full osteoclastic (OC) differentiation from multipotential hematopoietic precursor cells into mature multinucleated bone-resorptive OCs in the presence of the permissive factor macrophage colony-stimulating factor (M-CSF) (1-7). RANKL expressed on the surface of osteoblasts (OB) or bone marrow stromal cells (BMSC) interacts with a cell surface receptor, RANK, present on pre-OC (induced by M-CSF) and mature OC to stimulate their fusion, development, bone resorption, and cell survival (5-9). RANKL expression increases during early OB development and is up-regulated in OB and BMSC by various proresorptive stimuli such as parathyroid hormone (PTH), 1,25-dihydroxyvitamin D 3 (VD 3 ), dexamethasone (Dex), prostaglandin E 2 , or interleukin-11 (IL-11) (6, 10 -12). Recently, the pro-reso...

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 preOCs, 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-␣ 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 pitresorbing 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.

ABSTRACT

Nitric oxide synthase (NOS) isoenzymes generate nitric oxide (NO), a sensitive multifunctional intercellular signal molecule. High NO levels are produced by an inducible NOS (iNOS) in activated macrophages in response to proinflammatory agents, many of which also regulate local bone metabolism. NO is a potent inhibitor of osteoclast bone resorption, whereas inhibitors of NOS promote bone resorption both in vitro and in vivo. The possibility that osteoclasts, like macrophages, express a regulated iNOS and produce NO as a potential autocrine signal following inflammatory stimulation was investigated in well-characterized avian marrow-derived osteoclast-like cells. NO production (reflected by medium nitrite levels) was markedly elevated in these cells by the proinflammatory agents lipopolysaccharide (LPS) and the synergistic action of IL-la, TNFa, and IFNy. Inhibitors of NOS activity (aminoguanidine, L-NAME) or iNOS induction (dexamethasone, TGFP) reduced LPS-stimulated nitrite production. LPS also increased the NOS-associated diaphorase activity of these cells and their reactivity with anti-iNOS antibodies. RT-PCR cloning, using avian osteoclast-like cell RNA and human iNOS primers, yielded a novel 900 bp cDNA with high sequence homology (76%) to human, rat, and mouse iNOS genes. In probing osteoclast-like cell RNA with the PCR-derived iNOS cDNA, a 4.8 kb mRNA species was detected whose levels were greatly increased by LPS. Induction of iNOS mRNA by LPS, or by proinflammatory cytokines, occurred prior to the rise of medium nitrite in time course studies and was diminished by dexamethasone. Moreover, osteoclast-like cells demonstrated an upregulation of NO production and iNOS mRNA by IL-8 and IL-10, regulatory mechanism's not previously described. it is concluded that osteoclast-like cells express a novel iNOS that i s upregulated by inflammatory mediators, leading to NO production. Therefore, NO may serve as both a paracrine and autocrine signal for modulating osteoclast bone resorption. k 1996 Wiley-Liss, Inc

Osteoclast bone resorption is essential for normal calcium homeostasis and is therefore tightly controlled by calciotropic hormones and local modulatory cytokines and factors. Among these is nitric oxide (NO), a multifunctional free radical that potently inhibits osteoclast bone resorption in vitro and in vivo. Previous findings led us to propose that NO might serve as an autocrine, as well as paracrine, regulator of osteoclast function. This premise was investigated using isolated bone-resorptive avian osteoclasts and focusing on the inducible isoform of NO synthase (iNOS) responsible for inflammatory stimulated high-level NO synthesis in other cells. Avian osteoclasts expressed both iNOS messenger RNA (mRNA) and protein. However, inflammatory cytokines that induce iNOS mRNA, protein, and NO in other cells did not do so in avian osteoclasts, consistent with the known role of inflammatory stimuli in promoting osteoclast resorption and localized bone loss. In searching for potential modulators of osteoclast iNOS, protein kinase C activation [by phorbol 12-myristate 13-acetate (PMA)] and intracellular Ca2+ rises (A23187) were each found to elevate osteoclast iNOS mRNA and protein levels, while increasing NO release and reducing osteoclast bone resorption. The iNOS selective inhibitor aminoguanidine suppressed stimulated osteoclast NO production elicited by either signal, but reversed only the resorption inhibition due to raised Ca2+. Thus, whereas additional inhibitory signals are presumably coproduced in osteoclasts treated with PMA, osteoclast iNOS-derived NO may act as an autocrine signal to mediate Ca2+-inhibited bone resorption. These findings document for the first time an iNOS whose mRNA levels are regulated by Ca2+ or PMA, but not inflammatory stimuli, and the autocrine production of NO as a Ca2+ sensing signal to suppress osteoclast bone resorption. The unusual regulation of osteoclast iNOS makes it a potentially attractive target for designing novel therapeutic agents to alleviate excessive bone loss.