Lipopolysaccharide (LPS) modulates bone resorption by augmentation of osteoclastogenesis. It increases in osteoblasts the production of RANKL, interleukin (IL)-1, prostaglandin E 2 (PGE 2 ), and TNF-␣, each known to induce osteoclast activity, viability, and differentiation. We examined the role of direct interactions of LPS with osteoclast precursors in promoting their differentiation. To this end, we have used bone marrow mononuclear cell preparations in the absence of osteoblasts or stromal cells. We found that LPS does not induce osteoclast differentiation in these cells. Moreover, the inclusion of LPS blocked the osteoclastogenic activity of RANKL. However, LPS is a potent inducer of osteoclastogenesis in RANKL-pretreated cells, even if present in the absence of exogenous RANKL. Osteoprotegerin (OPG) does not affect the stimulatory phase of LPS modulation of osteoclastogenesis, ruling out involvement of endogenous RANKL. LPS induces the expression of TNF-␣ and IL-1␤ in osteoclast precursors, regardless if they were or were not pretreated with RANKL. These two cytokines induced osteoclast differentiation in RANKL-pretreated cells. To examine if these cytokines mediate LPS effect in an autocrine mechanism, we measured the effect of their neutralization on LPS osteoclastogenic activity. Although neutralization of IL-1␤ did not affect LPS activity, a marked inhibition was observed when TNF-␣ was neutralized. However, TNF-␣ expression was increased also in conditions in which LPS inhibited RANKL osteoclastogenic activity. We found that LPS reduces the expression of RANK and macrophage colony-stimulating factor (M-CSF) receptor. In summary, LPS impacts on osteoclastogenesis also via its interactions with the precursor cells. LPS inhibits RANKL activity by reducing the expression of RANK and M-CSF receptor and stimulates osteoclastogenesis in RANKL-pretreated cells via

HL-60, a cell line established from a patient with promyelocytic leukaemia, responds to a variety of inducing agents by ceasing division and acquiring some of the characteristics of either granulocytes or monocytes. Among the agents so far tested, only a comparative few occur naturally in vertebrates and would appear to have significant clinical potential in the treatment of leukaemic patients. One of the most promising of these is the dihydroxymetabolite of vitamin D3, 1,25(OH)2D3. This compound circulates in normal man and has a major role in calcium homeostasis. Moreover, it has recently been reported that 1,25(OH)2D3 increases the survival time of mice injected with myeloid leukaemia cells. We and McCarthy et al. have previously shown that HL-60 cells respond to near physiological levels of 1,25(OH)2D3 by rapidly acquiring a number of monocyte-like features. Here we document that these phenotypic changes are preceded by a marked decrement in the expression of the c-myc oncogene. In fact, the diminution in the level of c-myc mRNA parallels the dose dependency and metabolite specificity shown by the various other indicators of phenotypic change. In addition, we demonstrate that removal of vitamin D3, after the onset of maturational change, results in the reappearance of elevated myc mRNA levels. We believe this to be the first demonstration of a sequential relationship between the application of an exogenous inducing agent, a reduction in myc mRNA levels and the development of characteristics associated with normal cell maturation.

1,25-Dihydroxyvitamin D3 [1,25(0H)2D3] stimulates bone resorption in man and other vertebrates, in part, by increasing the number of osteoclasts, the principal resorbing cells of bone. Because osteoclasts are very likely derived from a member(s) of the mononuclear phagocyte family, we determined if 1,25(OH)2D3 promotes maturation of these cells by studying its effects on the human promyelocytic leukemia cell line HL-60. Of the vitamin D3 metabolites tested, only 1,25(OH)2D3, at 10-10 to 10-7 M, induces the differentiation of HL60 into mono-and multinucleated macrophage-like cells. Phenotypic change is evident within 24 hr and reaches a plateau between 72 and 96 hr of incubation. The changes are metabolite-specific and include (i) adherence to substrate, (ii) acquisition of the morphological features of mature monocytes, (iii) a 4-to 6-fold enhancement in lysozyme synthesis and secretion, (iv) increase in the fraction of a-naphthyl acetate esterase-positive cells from approximately 2% to 100% of the population, and (v) the acquisition of several monocyte-associated cell surface antigens. More importantly, treated HL-60 cells acquire the capacity to bind and degrade bone matrix, two of the essential, functional characteristics of osteoclasts and related boneresorbing cells. These results, considered together with the reported action of 1,25(OH)2D3 on nontransformed mononuclear cells, are consistent with the view that vitamin D3 enhances bone resorption and osteoclastogenesis in vivo by promoting the differentiation of precursor cells.Of the several circulating factors known to affect bone resorption, one of the most potent is 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. This compound, when administered in picomolar to nanomolar concentrations, markedly stimulates resorptive activity (1) and promotes a readily measurable increase in the number of osteoclasts, the principal resorbing cells of bone (2). It is generally assumed that the appearance of increased numbers of osteoclasts is responsible for enhanced bone resorption. However, the mechanism(s) by which 1,25(OH)2D3 alters the size of the osteoclast population, and therefore resorptive activity, is presently unknown.Osteoclasts originate by fusion of circulating mononuclear precursor cells and almost certainly represent one of the endstage cells of mononuclear phagocyte differentiation (3, 4). Like osteoclasts, other mature, nonproliferative members of this family-e.g., monocytes and macrophages (Mos)-possess the capacity to attach to and degrade bone matrix (5, 6), and therefore they serve as useful models with which to study the mechanisms of bone resorption in tissue culture. In a previous study, we showed that Mos isolated from vitamin D-deficient animals exhibit, in vitro, the same bone resorptive dysfunction characteristically observed in the intact, calciferol-deprived animal (7). This dysfunction is not corrected by the direct addition of 1,25(OH)2D3 to M4 cultures, but administration of the metabolite to vitamin D-depleted animals for several days ...

Osteoblasts or bone marrow stromal cells are required as supporting cells for the in vitro differentiation of osteoclasts from their progenitor cells. Soluble receptor activator of nuclear factor-kappaB ligand (RANKL) in the presence of macrophage colony-stimulating factor (M-CSF) is capable of replacing the supporting cells in promoting osteoclastogenesis. In the present study, using Balb/c-derived cultures, osteoclast formation in both systems-osteoblast/bone-marrow cell co-cultures and in RANKL-induced osteoclastogenesis-was inhibited by antibody to tumor necrosis factor-alpha (TNF-alpha), and was enhanced by the addition of this cytokine. TNF-alpha itself promoted osteoclastogenesis in the presence of M-CSF. However, even at high concentrations of TNF-alpha the efficiency of this activity was much lower than the osteoclastogenic activity of RANKL. RANKL increased the level of TNF-alpha mRNA and induced TNF-alpha release from osteoclast progenitors. Furthermore, antibody to p55 TNF-alpha receptors (TNF receptors-1) (but not to p75 TNF-alpha receptors (TNF receptors-2) inhibited effectively RANKL- (and TNF-alpha() induced osteoclastogenesis. Anti-TNF receptors-1 antibody failed to inhibit osteoclastogenesis in C57BL/6-derived cultures. Taken together, our data support the hypothesis that in Balb/c, but not in C57BL/6 (strains known to differ in inflammatory responses and cytokine modulation), TNF-alpha is an autocrine factor in osteoclasts, promoting their differentiation, and mediates, at least in part, RANKL's induction of osteoclastogenesis.

Osteoclasts are multinucleated cells that derive from hematopoietic progenitors in the bone marrow which also give rise to monocytes in peripheral blood, and to the various types of tissue macrophages. Osteoclasts are formed by the fusion of precursor cells. They function in bone resorption and are therefore critical for normal skeletal development (growth and modeling), for the maintenance of its integrity throughout life, and for calcium metabolism (remodeling). To resorb bone, the osteoclasts attach to the bone matrix, their cytoskeleton reorganizes, and they assume polarized morphology and form ruffled borders to secrete acid and collagenolytic enzymes and a sealing zone to isolate the resorption site. Identification of the osteoclastogenesis inducer, the receptor activator of nuclear factor-kappaB ligand (RANKL), its cognate receptor RANK, and its decoy receptor osteoprotegerin (OPG), has contributed enormously to the dramatic advance in our understanding of the molecular mechanisms involved in osteoclast differentiation and activity. This explosion in osteoclast biology is reflected by the large number of reviews which appeared during the last decade. Here I will summarize the "classical" issues (origin, differentiation, and activity) in a general manner, and will discuss an untouched issue (multinucleation) and a relatively novel aspect of osteoclast biology (osteoimmunology).

Regulation of osteoclastogenesis by lipopolysaccharide (LPS) is mediated via its interactions with toll-like

The interplay between the immune system and bone metabolism has been recognized as important for both of these systems. Various factors produced and released during immune responses markedly affect bone cells and bone metabolism. Meanwhile, niches for lymphocytes in bone also play an important role in the biology of these cells. Osteoimmunology, a new area of research focusing on associations between the immune and bone systems, is based on the concept that deeper investigation of the relationships between these systems will enhance our understanding of their biology and contribute to the discovery of novel therapeutic approaches for diseases of the two systems. Toll-like receptors (TLRs), the focus of this review, sense pathogen-derived molecules and initiate the inflammatory reactions of innate immune cells. TLRs are also expressed in bone cells, and their activation affects osteoclast differentiation and activity in a complex manner: TLR activation in early osteoclast precursors blocks the differentiation of those cells, while in cells that have already started their osteoclastic differentiation, it stimulates this process and increases the survival rates of mature osteoclasts (OCs). Activation of TLRs in osteoblasts (OBs) induces the production of osteoclastogenic cytokines, such as RANKL and TNF-alpha, thereby contributing to TLR ligand-induced osteoclastogenesis. These processes are the reason for the bone loss observed in variety of infectious diseases. The inhibition of osteoclastogenesis by TLR activation in early precursor cells may play a role in reducing the excessive bone loss caused by pathogenic infection and shifting the balance between the bone and immune systems during infection to recruit immune cells.

The article to which this erratum refers was published in J Cell Biochem (2007) 102: 1130–1139. © 2007 Wiley‐Liss, Inc.