JBJS | Current Concepts Review - Cellular Biology of Bone-Resorbing Cells*

The Journal of Bone & Joint Surgery, Volume 78, Issue 7

Current Concepts Review | July 01, 1996

Current Concepts Review - Cellular Biology of Bone-Resorbing Cells*

N. A. ATHANASOU, PH.D., F.R.C.PATH†, OXFORD, ENGLAND

*No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. Funds were received in total or partial support of the research or clinical study presented in this article. The funding sources were: The Wellcome Trust, The Cancer Research Campaign, and The British Orthopaedic Association Wishbone Trust.

The Journal of Bone & Joint Surgery. 1996; 78:1096-1112

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Introduction

Introduction | Criteria for Recognition and Definition of Osteoclasts | Cell Lineage of Osteoclasts | Cellular and Hormonal Control of Differentiation and Function of Osteoclasts | Cellular and Hormonal Mechanisms Associated with Pathological Bone Resorption | References

Resorption of bone occurs continuously throughout life, first as part of skeletal growth and modeling and, later, in the process of bone-remodeling in the adult skeleton. Since the 1970's, considerable progress has been made in unraveling the basic cellular mechanisms that regulate the formation and activity of the osteoclast, the main cellular agent of bone resorption. These recent advances have resulted mainly from the introduction of new methods of isolation and culture of osteoclasts and evaluation of resorptive activity; techniques have also been developed for the generation of osteoclasts in long-term culture of precursor cells found in hematopoietic tissues and peripheral blood. Although reports have suggested that other cells (such as tumor cells or macrophages) are capable of degrading bone matrix^48,104,152 and releasing local factors that contribute to resorption of bone^{45,102,105,113}, the osteoclast is the only cell specialized for this function.

The purpose of this review is to integrate new knowledge regarding the formation of osteoclasts and the regulation of their activity within a general view of the cellular biology of normal and pathological bone resorption. After a review of the criteria by which osteoclastic cells are defined, the origin and formation of osteoclasts will be considered, along with mechanisms governing their recruitment, activation, and function. The manner in which these mechanisms contribute to osteoclastic bone resorption under various pathological conditions as well as the possible roles of other cells (both skeletal and extraskeletal in origin) in this process will be discussed.

†Department of Pathology, Nuffield Orthopaedic Centre, Headington, Oxford OX3 7LD, England.

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TABLE I DEFINING CRITERIA OF OSTEOCLASTS AND THEIR PRESUMED PRECURSORS

*The findings are osteoclast-specific.

Morphological

Light Microscopy

Multinuclearity

Location at sites of bone resorption

Transmission electron microscopy*

Ruffled borders

Clear zones

Enzyme histochemistry

Expression of tartrate-resistant acid phosphatase

Expression of tartrate-resistant trinucleotide phosphatase

Expression of type-II carbonic anhydrase isoenzyme

Immunohistochemistry/immunology

Expression of restricted range of leukocyte or macrophage-associated antigens (CD13, CD15, CD45, CD54, CD68, and CD51 [vitronectin receptor])

No expression of CD11/18, CD14, HLA-DR, Fc and C3b receptor

Functional behavior*

Ability to form resorption lacunae on a bone substrate

Response to calcitonin

Ability to bind calcitonin (possesses calcitonin receptors)

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TABLE I DEFINING CRITERIA OF OSTEOCLASTS AND THEIR PRESUMED PRECURSORS

*The findings are osteoclast-specific.

Morphological

Light Microscopy

Multinuclearity

Location at sites of bone resorption

Transmission electron microscopy*

Ruffled borders

Clear zones

Enzyme histochemistry

Expression of tartrate-resistant acid phosphatase

Expression of tartrate-resistant trinucleotide phosphatase

Expression of type-II carbonic anhydrase isoenzyme

Immunohistochemistry/immunology

Expression of restricted range of leukocyte or macrophage-associated antigens (CD13, CD15, CD45, CD54, CD68, and CD51 [vitronectin receptor])

No expression of CD11/18, CD14, HLA-DR, Fc and C3b receptor

Functional behavior*

Ability to form resorption lacunae on a bone substrate

Response to calcitonin

Ability to bind calcitonin (possesses calcitonin receptors)

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Fig. 1. The osteoclast cell-lineage pathway. The asterisk indicates bone-marrow and mononuclear phagocyte cell populations from which multinucleated calcitonin receptor-positive and lacunar bone-resorbing cells have been generated. The osteoclast forms part of the mononuclear phagocyte system, being derived from the pluripotential hematopoietic stem cell and sharing a common bone-marrow progenitor with monocytes and macrophages. The mononuclear osteoclast precursor expresses a monocyte/macrophage phenotype and circulates in the monocyte fraction of peripheral blood. In the microenvironment of bone, under the influence of osteoblasts, these precursors acquire the phenotypic characteristics of osteoclasts and differentiate to form mature osteoclasts capable of forming resorption lacunae; macrophage colony-stimulating factor and 1,25-dihydroxyvitamin D3 are required for this to occur. Systemic hormones and local humoral factors influence not only the osteoblastic control of the formation of osteoclasts but also the osteoblastic modulation of osteoclastic bone-resorbing activity.

Criteria for Recognition and Definition of Osteoclasts

Studies on the biology of bone-resorbing cells can be properly evaluated only if the criteria for the recognition of osteoclasts are clearly defined. On histological sections, osteoclasts are readily identified as large multinucleated cells that lie in apposition to a bone surface undergoing lacunar resorption (resorption in Howship lacunae); however, they and their mononuclear precursors are not so easily identified in heterogeneous cell populations recovered from bone and hematopoietic tissues. Both in situ and in vitro, it has proved difficult to distinguish osteoclasts from other cells, particularly macrophages and multinucleated giant cells, termed macrophage polykaryons, which are the products of macrophage fusion¹⁴⁴.

In morphological terms, multinuclearity is not sufficient to distinguish osteoclasts from macrophage polykaryons. There is also uncertainty about the cellular identity of multinucleated cells found in giant-cell tumors and some inflammatory lesions; these osteoclast-like giant cells have been shown to lack many phenotypic features of the osteoclast and yet to be capable of forming resorption lacunae in a manner similar to that of osteoclasts^11,13,17,19. Ultrastructurally, osteoclasts are distinguished by the presence of a ruffled border, a complex system of villous folds of the plasma membrane beneath which bone resorption occurs; this is surrounded by a clear zone, which anchors the osteoclast to the bone surface⁶¹. Similar morphological features have been noted on mononuclear cells found near osteoclasts in bone, and these cells have been designated preosteoclasts^127,137. Macrophages and macrophage polykaryons show similar convolutions of the membrane under conditions associated with increased cellular activity^2,144, but they do not possess such complex membrane structures. Transmission electron microscopy, however, is too laborious to be used for routine identification. It should be appreciated that osteoclasts elaborate a ruffled border only on that part of their membrane surface that is in contact with bone, and it is difficult, if not impossible, to demonstrate ruffled borders satisfactorily in cultures of cells. Thus, while the absence of a ruffled border has been often cited as proof that an isolated cell population is not osteoclastic, identification of such a border may not be a reliable way to determine in vitro if a multinucleated cell is an osteoclast.

Because osteoclasts are rich in certain enzymes, the enzyme histochemistry is commonly used to identify osteoclasts. Osteoclasts are particularly rich in the acid phosphatase isoenzyme, tartrate-resistant acid phosphatase^100,160. Tartrate-resistant acid phosphatase is a convenient cytochemical marker of osteoclasts, but it is not osteoclast-specific; it has been demonstrated in osteoblasts²⁴ and in cells in many extraosseous tissues, such as alveolar and activated tissue macrophages^9,47. Its use to identify osteoclasts in vitro is also limited by the fact that monocytes and tissue macrophages are known to become positive for tartrate-resistant acid phosphatase after several days in culture^66,101. Tartrate-resistant acid phosphatase and other enzyme markers such as tartrate-resistant trinucleotide phosphatase and carbonic anhydrase isoenzymes should be regarded as osteoclast-associated rather than osteoclast-specific^8,9,111. When these enzymes are not expressed, however, they are useful as negative markers to show that a cell population does not contain osteoclasts or committed osteoclast precursors.

The development of several monoclonal antibodies to macrophage and osteoclast cell-surface antigens has also been used to identify osteoclasts and their precursors^{71,72,112,115}. Osteoclasts have been shown to highly express some cell-surface antigens, such as the a-chain of the vitronectin receptor (CD51), one of the integrin family of glycoprotein membrane antigens that are involved in both cell-cell and cell-matrix interactions^71,72. However, there has been no convincing evidence of a cell-surface antigen that is osteoclast-specific, as many of these antigens have also been found on other cells, including macrophages and macrophage polykaryons¹⁸. Like macrophages and macrophage polykaryons, osteoclasts strongly express CD45, the leukocyte common antigen¹⁴, and a number of other macrophage-associated antigens^10,15, including CD13, CD15, CD68, and CD54. However, osteoclasts do not express multiple other macrophage-associated antigenic markers^10,15, such as CD11a, CD11b, CD14, CD18, and HLA-DR, as well as receptors for Fc and complement components¹³⁸. Knowledge of the antigenic phenotype expressed by human osteoclasts is useful in that it provides a range of positive and negative markers whereby these cells can be distinguished from macrophages and macrophage polykaryons¹⁰. Apart from their utility in distinguishing osteoclasts and their precursors, techniques involving monoclonal antibodies have also shown that osteoclasts and their likely progenitors possess cell-surface receptors that are likely to be important in cell-cell and cell-matrix interactions involved in the recruitment and cellular fusion of osteoclast precursors and in the attachment of osteoclasts to bone matrix⁷⁰.

The two most widely accepted means of specifically identifying an osteoclast are the demonstrations of expression of calcitonin receptors and of lacunar bone resorption. Osteoclastic activity is directly and specifically inhibited by calcitonin³⁴, and the demonstration of receptors that bind calcitonin is considered to be a reliable and highly specific marker of a mammalian osteoclast^67,109. Avian osteoclasts generally lack calcitonin receptors and do not appear to respond to calcitonin¹⁰⁸. In contrast, the unique ability of the osteoclast to form resorption lacunae in bone provides a specific and reliable way of identifying this cell in any species of vertebrate. This is best demonstrated by scanning electron microscopy, with which osteoclasts can be seen to begin forming resorption lacunae on a mineralized substrate, such as bone or dentin, almost immediately after they have been isolated from bone^26,39. Other cell types, including monocytes and macrophages, are capable of dissolution of both the inorganic and the organic matrix components of bone but without the formation of lacunae^104,152; therefore, it is essential that dissolution of bone matrix be shown to be lacunar to be certain that bone resorption is due to fully differentiated osteoclasts or a population of cells committed to osteoclastic differentiation. The recognition of osteoclasts is thus based on morphological, histochemical, immunological, and functional criteria (Table I).

Cell Lineage of Osteoclasts

Osteoclasts are commonly found in large numbers at sites of active bone resorption in the developing skeleton and in pathological conditions characterized by extensive osteolysis. Accordingly, in addition to investigating factors controlling the resorptive activity of osteoclasts, much recent research has been directed toward defining the cell lineage of osteoclasts and the factors that determine the origin, recruitment, and formation of osteoclasts found at sites of bone resorption.

Amitotic and mitotic division by osteoclasts has not been seen, and it is generally accepted that these multinucleated cells form only through the fusion of their mononuclear precursors⁶¹. This process occurs rapidly and is subject to systemic hormonal control^21,25. In the past, osteoclasts and osteoblasts were thought to arise from a single precursor cell¹⁵⁷, but it is now generally accepted that osteoclasts are formed by the fusion of circulating mononuclear precursor cells, which are themselves of hematopoietic origin. Evidence to support this conclusion has come from several experimental approaches. With use of parabiosis—the establishment of a common circulation between two rats, one normal and one in which all of the hematopoietic tissues and local precursors had been destroyed by irradiation—Gothlin and Ericsson⁶⁰ showed that the cells that fused to form osteoclasts in a healing fracture produced in the irradiated rat must have been derived from the non-irradiated rat through the circulation. In quail-chick chimeras in which the long bones of one species had been grafted onto the chorioallantoic membrane of another, it was possible to exploit the known morphological difference between chick and quail nuclei to determine the origin of different types of cells present in the developing bone^77,78; in this way, it was shown that osteoclasts formed by fusion of host mononuclear cells derived from the vasculature of the chorioallantoic membrane, whereas the origin of the osteoblasts was the local donor graft. The inherited bone disease osteopetrosis has also provided a useful model with which to study the ontogeny of osteoclasts. Osteopetrosis is a group of skeletal disorders characterized by sclerosis of bone as a result of deficient osteoclastic bone resorption^97,118. Both parabiotic union of osteopetrotic mice with normal littermates and transplantation of hematopoietic stem-cell suspensions from normal littermates to affected offspring have been shown to cure some forms of this disease^163-165; this indicates that precursors of osteoclasts are of hematopoietic derivation and are present in the circulation. This has also been confirmed in humans, as transplantation of bone marrow has been successful in the treatment of infantile malignant osteopetrosis⁴⁰; karyotypic analysis of osteoclasts formed in patients in whom bone marrow had been transplanted confirmed that these cells were of donor origin.

Virtually all subsequent studies^{7,14,22,89,158} have shown that osteoclasts form by fusion of circulating cells of hematopoietic origin and that this is the only means whereby these cells are formed. The lineage of osteoclasts is thus distinct from that of the connective-tissue cells of bone and the bone-marrow stroma (osteoblasts, chondrocytes, adipocytes, bone-marrow stromal cells, and fibroblasts). These connective-tissue cells are derived from a separate primordial mesenchymal stem cell of bone from which more committed progenitors for each of the aforementioned cells of bone are produced¹¹⁶. This is currently an area of active research and it is not certain how each of these stromal cell lines is related, although there is some evidence that osteoblastic and chondroblastic cells are derived from a single cell lineage that, depending on extracellular conditions, follows a chondrogenic or osteogenic pathway of differentiation²⁰.

After it was established that the osteoclast was of hematopoietic origin and had a circulating (presumably mononuclear) precursor, it was expected that the monocyte would represent that precursor cell³⁰. Like other cells of the mononuclear phagocyte system, osteoclasts are avidly phagocytic cells that show trypsin-resistant adherence to a glass or plastic substrate²⁹; they share many morphological, cytochemical, and immunophenotypic characteristics with monocytes and macrophages, some of which have already been mentioned. Peripheral blood monocytes are known to be chemotactically attracted to resorbed products of the bone matrix^103,106 and to fuse readily to form multinucleated cells^49,144. Monocytes have been observed to fuse with multinucleated cells in cultures of isolated avian osteoclasts¹⁶⁹, and labeled particles incorporated into monocytes and macrophages have also been found in osteoclasts^75,156. Baron et al.²² showed that mononuclear cells containing non-specific esterase, a macrophage marker, appear at sites of bone resorption before mononuclear cells that contain either tartrate-resistant acid phosphatase or both tartrate-resistant acid phosphatase and non-specific esterase; this suggests that the mononuclear osteoclast precursor cell that fuses to form multinucleated osteoclasts is a cell of the monocyte-macrophage lineage. Although monocytes and macrophages are known to be capable of degrading both the organic and the inorganic matrix components of bone particles^104,152, unlike osteoclasts they are not capable of lacunar bone resorption³³; this has been noted even in the presence of 1,25-dihydroxyvitamin D₃, which is known to promote the fusion of monocytes and macrophages, their adhesion to substrates, their phagocytic activity, and their ability to degrade bone particles^1,23,156. 1,25-dihydroxyvitamin D₃ also has been found to be an absolute requirement for the formation of osteoclasts from osteoclast precursor cell populations in hematopoietic tissue and peripheral blood in vitro^121,148,158. Unlike osteoclasts, monocytes, macrophages, and macrophage polykaryons do not respond to calcitonin or express calcitonin receptors¹⁰⁹; they also do not express the specific ultrastructural features of osteoclasts when they are cultured alone on a mineralized substrate⁷⁹.

In the absence of convincing evidence that mature cells of the mononuclear phagocyte system (monocytes and macrophages) directly fuse to form osteoclasts, and on the basis of the knowledge that osteoclasts contain many functional and other characteristics that are sufficiently unique to distinguish them from other cells of hematopoietic origin, it was proposed that the osteoclast is the final product of a specialized line of hematopoietically derived osteoclast progenitors, originating from either a unique primordial stem cell or an early osteoclast-specific derivative of the pluripotential hematopoietic stem cell³². This early progenitor would be equivalent to that of other major products of the hematopoietic stem cell (committed precursors for erythrocytes, platelets, and granulocytes and monocytes). This hypothesis was originally based in part on several criteria for defining osteoclasts, such as their immunophenotype and histochemical profile, which, as already noted, have not proved to be specific for these cells. It was therefore not surprising when it was subsequently found that osteoclasts could be generated from highly purified pluripotent hematopoietic stem cells^63,135,136 as well as from more committed hematopoietic and circulating cell populations^{7,68,89,147,153,158}. It has also been shown that leukocyte common antigen is present on human osteoclasts¹⁴; this antigen is expressed by all nucleated progeny of the pluripotential hematopoietic stem cell, indicating that human osteoclasts (and their circulating precursors) are derived from the same stem cell that other peripheral blood leukocytes are derived from.

Various experimental systems have been devised whereby osteoclasts can be induced to differentiate in vitro from both committed hematopoietic progenitor and circulating monocyte and tissue macrophage populations. This work has provided important information not only on the nature of the precursor populations that form osteoclasts but also on the cellular and hormonal conditions that are necessary for this to occur. In the metatarsal culture system developed by Burger et al.²⁸, various candidate precursor cell populations were cultured with stripped rudiments of bone, devoid of osteoclast progenitors, from fetal mice. In this system, it was shown that osteoclasts formed when these rudiments of bone were maintained in long-term culture with murine marrow mononuclear-cell fractions, composed of colony-forming units for granulocytes and macrophages, and immature precursors for mononuclear phagocytes but not mature tissue macrophages^28,153. The observation that osteoclasts and mononuclear phagocytes share a common marrow progenitor such as the colony-forming units for granulocytes and macrophages has been further supported by the results from long-term avian and mammalian hematopoietic (bone-marrow or spleen) culture systems^7,89,147,148. Kurihara et al.⁸⁹, using a human bone-marrow culture system, showed that, in the presence of 1,25-dihydroxyvitamin D₃, osteoclast-like multinucleated cells formed from colony-forming units for granulocytes and macrophages. Takahashi et al.¹⁴⁶ came to a similar conclusion and, using long-term murine marrow and spleen culture systems, they made the important observation that osteoblastic or other specific bone-derived stromal cells are an absolute requirement for the differentiation of osteoclast precursors of hematopoietic origin into functional osteoclasts.

Hematopoietic tissue contains a mix of progenitor and stromal cell populations, and thus it is difficult to identify with certainty the characteristics of the specific cell population that represents osteoclast progenitors. Mononuclear-cell fractions in peripheral blood can be more readily separated and characterized. The capacity of murine peripheral blood monocytes to form osteoclasts was investigated with use of a co-culture system similar to that employed with marrow or spleen cells; it was found that bone-resorbing osteoclasts positive for tartrate-resistant acid phosphatase and calcitonin receptors could be generated efficiently¹⁵⁸. Murine mononuclear phagocyte populations of extraskeletal tissue origin (alveolar macrophages) were also found to be capable of osteoclastic differentiation under these conditions. These cells were co-cultured with the preadipocytic bone-marrow stromal cell line ST2 in the presence of 1,25-dihydroxyvitamin D₃ and glucocorticoids. It was also found that, when the mononuclear cells in the peripheral blood were fractionated into two cell populations, one monocyte-depleted and one monocyte-enriched, the monocyte-depleted population failed to form colonies on ST2 cell layers, whereas the monocyte-enriched population formed several colonies positive for tartrate-resistant acid phosphatase. Quinn et al.¹²¹ confirmed these findings using co-cultures of murine monocytes and UMR 106 osteoblast-like cells. They found that the differentiation of osteoclasts from mononuclear phagocytes could be inhibited by calcitonin and prostaglandins and that it resulted when as few as 100 peripheral blood monocytes were co-cultured with UMR 106 cells; this indicated that the proportion of cells in the monocyte fraction capable of osteoclastic differentiation was surprisingly high and was an argument against such isolated cells being a population of specialized predetermined circulating osteoclast precursors. It has also been shown that when macrophages from mice, rats, and humans were isolated from various normal and abnormal (neoplastic and inflammatory) tissues, as well as macrophage cell lines, the macrophages were also capable of osteoclastic differentiation under the same cellular and hormonal conditions of culture^{12,120,122-124,139}.

The formation of osteoclasts from isolated hematopoietic cells of the colony-forming unit for granulocytes and macrophages as well as from isolated monocyte and macrophage cell populations provides substantial proof that the osteoclast is a member of the mononuclear phagocyte system; these results show that osteoclasts, monocytes, and macrophages are derived from the same hematopoietic precursor-cell population and that the mononuclear precursor cells that differentiate into osteoclasts circulate in the monocyte fraction of peripheral blood (Fig. 1). These studies also make it clear that these osteoclast precursors do not demonstrate the osteoclastic characteristics of expression of tartrate-resistant acid phosphatase and calcitonin receptors and the ability to carry out lacunar bone resorption; in addition, multinucleated cells exhibiting all of the aforementioned osteoclast-specific characteristics were formed only when these cells were cultured in contact with osteoblasts or other bone-marrow stromal cells^121,158. Thus, by all current criteria, osteoclast precursors should be regarded as mononuclear phagocytes that express monocyte or macrophage markers. The failure of previous studies to demonstrate that bone-resorbing osteoclasts could form in vitro from various isolated mononuclear phagocyte cell populations was due to the fact that an essential bone-marrow stromal-cell element was not included in the cultures³³.

It is notable that in co-cultures of bone-marrow stromal cells and osteoclast precursors of hematopoietic or of monocyte or macrophage origin, lacunar bone-resorbing cells form only after several days of incubation. This process appears to involve a number of steps associated with the proliferation and differentiation of mononuclear osteoclast precursor cells into mature functional osteoclasts. Tanaka et al.¹⁵¹ used autoradiography with ³Hydrogen-thymidine and showed that, in a co-culture system of murine osteoblasts and spleen cells, mononuclear osteoclast precursors primarily proliferated during the first four days of culture and then, in the last two days of culture, differentiated into osteoclastic cells; hydroxyurea inhibited the formation of multinucleated cells when it was added to co-cultures in the first four days but not when it was added only in the final two days. These authors also found that the proliferation of osteoclast precursors was stimulated strongly by the macrophage colony-stimulating factor and less strongly by the granulocyte-macrophage colony-stimulating factor and by interleukin-3. Rapid and dramatic changes in the cytochemical features, the expression of calcitonin receptors, the antigenic qualities, and the functional phenotype of murine osteoclast precursors were found to accompany this process. Takahashi et al.¹⁴⁶ showed that murine osteoclast precursor cells of hematopoietic origin express macrophage-associated antigens that are rapidly lost when these precursors are co-cultured with bone-marrow stromal cells; they found that the number of cells expressing calcitonin receptors reached a maximum forty-eight hours after the establishment of the co-cultures, and the first evidence of bone resorption was seen soon after this. Moreover, they showed that, at one stage of differentiation, osteoclast precursors expressed macrophage markers, such as non-specific esterase and Mac-1 and Mac-2, as well as osteoclast markers, such as tartrate-resistant acid phosphatase and calcitonin receptors. These results are in agreement with those reported by Baron et al.²², who found cells showing cytochemical features of both macrophages and osteoclasts at sites of bone resorption. Thus, it would appear that the formation of osteoclasts from mononuclear phagocytes occurs in a number of well defined steps, each of which is associated with the loss and acquisition of specific phenotypic markers for macrophages and osteoclasts, respectively. It also implies that, in pathological situations in which osteoclasts form rapidly, such as after administration of parathyroid hormone²⁵, mononuclear phagocyte osteoclast precursors, primed and activated for the final steps of osteoclastic differentiation, are present in bone. Such mononuclear phagocytes are not unique to bone, as shown by the fact that macrophages isolated from extraskeletal tissues can be induced to form osteoclasts.

This process of differentiation of osteoclasts from cells that are found in isolated monocyte and extra-skeletal tissue macrophage cell populations needs to be understood in the context of the mononuclear phagocyte system; this includes not only cells that are highly efficient at phagocytosis but also a wide range of tissue-specific cell types that perform several highly specialized functions (for example, microglial cells, Kupffer cells, synovial lining cells, osteoclasts, and Langerhans cells)⁵⁹. Two theories have been proposed to account for this profound heterogeneity being the product of a single cell lineage⁹⁹. The first theory is that this diversity is hematopoietically programmed. Committed cell lines for the various tissue-specific cell types that make up the mononuclear phagocyte system are thought to be present in the bone marrow; from each of these cell lines, specific precursors that home only to a particular tissue are then released into the circulation. In the case of bone, it is thought that an osteoclast-specific cell line is formed at some point after the formation of the colony-forming unit for granulocytes and macrophages; the products of this cell line would be released into the circulation as specific mononuclear osteoclast precursors, which would then localize to bone as and when required. The alternative hypothesis is that there is a single highly dynamic monocyte-macrophage cell lineage and that the various specialized cell types that make up the mononuclear phagocyte system are the result of phenotypic alterations that occur when peripheral blood monocytes leave the circulation to become macrophages. Tissue macrophages have a long life span of months or years, and it is believed that modulating influences present in a specific local tissue microenvironment induce differentiation of the macrophage toward one of the specialized cell types of the mononuclear phagocyte system. In the case of bone, macrophages derived from peripheral blood monocytes that have left the bone vasculature would differentiate into fully mature osteoclasts under the influence of local cellular and humoral factors present within the microenvironment of bone.

This latter hypothesis is strongly supported by the work of Quinn et al.¹²¹, who showed that, in a co-culture system of murine peripheral blood leukocytes and osteoblastic cells, a surprisingly high proportion of the leukocytes in the circulation of the normal mouse were capable of osteoclastic differentiation. The formation of osteoclasts, which are capable of the highly specialized function of bone resorption, from extraskeletal cell populations of macrophages that were similarly cultured also strongly favors this hypothesis^122,158. These results, however, do not entirely exclude the possibility that the osteoclast precursors represent a subset of committed mononuclear osteoclast precursors that express only known monocyte or macrophage markers. In fact, it should be recognized that, in any cell population of monocytes and macrophages, there is considerable heterogeneity in terms of ultrastructural morphology, surface protein expression, cytochemical features, and functional behavior⁸⁸. All monocyte and macrophage populations are known to contain responsive cells that change their phenotype when they are activated by specific cellular, matrix, or cytokine signal³. Moreover, as much as 5 per cent of tissue macrophages are capable of proliferation¹⁶¹; these cells are indistinguishable morphologically from mature monocytes and macrophages. As a similar percentage of isolated monocytes and macrophages appear to be capable of undergoing osteoclastic differentiation in vitro, the osteoclasts may be formed from this fraction of relatively immature monocytes and macrophages. These cells do not begin to acquire the phenotypic characteristics of osteoclasts until they are placed in the cellular, hormonal, and tissue microenvironment of bone, where they undergo proliferation and differentiation into mature, functional osteoclasts.

Cellular and Hormonal Control of Differentiation and Function of Osteoclasts

Osteoclastic bone resorption does not occur randomly in the skeleton; instead, it occurs at sites where skeletal remodeling and restructuring are necessary. Therefore, a mechanism is needed whereby circulating osteoclasts and their precursors can localize to these sites. As already noted, osteoblasts and other bone-marrow stromal cells have an important role in the formation of osteoclasts. The manner in which these cells form a continuous network throughout bone indicates that they also have a regulatory role in the localization of osteoclasts. Mineral crystals are avidly ingested by mononuclear phagocytes, and constituents of the bone matrix, such as osteocalcin or type-I collagen peptides, are known to be chemotactic for monocytes^90,95,103. Osteoclast precursors, either already present in bone or emigrating from the bloodstream, are probably attracted to sites where resorption of bone will occur and are induced to mature by resident cells in bone. Osteoblasts are known to have the capacity to modulate the expression of adhesion molecules in the extracellular matrix that interact with integrins on osteoclasts; in this way, they may guide the localization and recruitment of osteoclasts and osteoclast precursors to sites needing resorption of bone or they may be involved in the initiation of resorption⁷⁰.

A major clue to the role of osteoblasts and other stromal cells in the differentiation of osteoclasts was found by studying the inherited op/op variant of murine osteopetrosis. In this condition, the number of osteoclasts is decreased, with a consequent decrease in resorption of bone and an increase in bone mass. The cause of this defect was found to be a failure of hematopoietic stromal cells to release functionally active macrophage colony-stimulating factor, due to an extra thymidine insertion at base pair 262 in the coding region of the gene for macrophage colony-stimulating factor¹⁶⁸. The administration of recombinant human macrophage colony-stimulating factor restored the number of osteoclasts and macrophages and cured the bone-resorption defect in op/op mice⁵⁰. At the same time, it was shown that osteoblastic cells obtained from op/op mice did not support osteoclastic differentiation in co-culture with normal spleen cells unless macrophage colony-stimulating factor and 1,25-dihydroxyvitamin D₃ were added to the co-cultures⁵¹. These findings provide strong evidence that the production of macrophage colony-stimulating factor by osteoblasts is critical for the development of osteoclasts. As noted earlier, macrophage colony-stimulating factor appears to be essential for both the proliferation and the differentiation of osteoclast progenitors¹⁵¹; it also appears to prolong the survival of mature osteoclasts⁵⁴. Macrophage colony-stimulating factor acts through c-fms, the macrophage colony-stimulating factor receptor, and its signal transduction is mediated by tyrosine kinase. Restoration of the macrophage population in the bone marrow of op/op mice after treatment with macrophage colony-stimulating factor is also consistent with the origin of osteoclasts being a resident mononuclear phagocyte population^50,51. However, although osteopetrotic mutations in mice provide valuable experimental models with which to study the development and function of osteoclasts, they do not strictly correspond to human osteopetrosis. This is evidenced by the finding that circulating macrophage colony-stimulating factor is not reduced in infantile malignant osteopetrosis in humans¹¹⁴.

Other growth factors (glycoprotein products that stimulate the proliferation of progenitor cells) and cytokines (polypeptide products of one cell that act locally on another) have also been reported to modulate the formation of osteoclasts. These include interleukin-1, interleukin-6, interleukin-11, leukemia inhibitory factor, and tumor necrosis factor^96,142,143. Unlike macrophage colony-stimulating factor, these peptide-signaling molecules do not appear to be essential cofactors for the formation of osteoclasts but, like macrophage colony-stimulating factor, their effects on the formation of osteoclasts necessitate the presence of osteoblastic or bone-marrow stromal cells. Interleukin-6 acts synergistically with interleukin-3 to stimulate the development of colony-forming units for granulocytes and macrophages, which, as noted earlier, contain the hematopoietic precursors of osteoclasts⁸⁸. Interleukin-6 is a multifunctional, locally acting cytokine that exerts its influence on cells through a cell-surface receptor made up of two components: a membrane-bound interleukin-6 receptor and a signal-transducing 130-kilodalton glycoprotein. When the interleukin-6 receptor is occupied by interleukin-6, the ligand-receptor complex binds the 130-kilodalton glycoprotein, which then transduces interleukin-6 signals¹⁴³. Interleukin-6 is not thought to stimulate the formation of osteoclasts from precursors alone but, rather, in combination with its soluble receptor¹⁴⁹. The soluble interleukin-6 receptor is formed by limited proteolysis of the cell membrane-bound interleukin-6 receptor and is present in the normal circulation (in greater amounts than interleukin-6 itself, in fact)¹⁴⁵. As osteoclasts are formed only when the soluble interleukin-6 receptor is added with interleukin-6 to co-cultures of bone-marrow precursors and osteoblasts that are in contact with each other, it is thought that cellular interaction between osteoblastic cells and osteoclastic progenitors is necessary for interleukin-6 to stimulate the formation of osteoclasts in bone.

Other cytokines that influence the differentiation of osteoclasts, such as interleukin-11 and leukemia inhibitory factor, also utilize the 130-kilodalton glycoprotein as a signal transducer¹⁴³. Interleukin-11 appears to be a major controlling factor in the formation of osteoclasts⁵⁶. It is produced by bone-marrow stromal cells and induces formation of osteoclasts in co-cultures of murine bone marrow and osteoblasts. The addition of neutralizing antibody to interleukin-11 also suppresses the formation of osteoclasts induced by 1,25-dihydroxyvitamin D₃, parathyroid hormone, interleukin-1, and tumor necrosis factor⁵⁶. Parathyroid hormone and 1,25-dihydroxyvitamin D₃ also increase the production of interleukin-11 in these cultures, and induction of osteoclast formation by interleukin-11 is prevented by blocking the synthesis of prostaglandins in these cultures.

Systemic hormonal factors, such as parathyroid hormone and parathyroid hormone-related peptide, and local factors, such as prostaglandins, also act through osteoblasts and bone-marrow stromal cells to stimulate the formation of osteoclasts and resorptive activity. Lorenzo et al.⁹¹ used autoradiography with ³Hydrogen-thymidine and showed that parathyroid hormone stimulates the recruitment of osteoclast precursors. Takahashi et al.¹⁴⁸ performed experiments using the murine bone-marrow system and concluded that parathyroid hormone induces the differentiation of immature to mature osteoclast precursors as well as their fusion to form multinucleated cells. Parathyroid hormone, parathyroid hormone-related peptide, and prostaglandins also require the presence of specific osteoblastic or bone-marrow stromal cells to achieve a stimulatory effect on the formation of osteoclasts from precursors of hematopoietic (spleen) origin^4,6,142,143. All of these agents appear to act through a mechanism mediated by cyclic adenosine 3',5'-monophosphate and protein kinase A^4,6,80. Prostaglandin E₂ also has been reported to be involved^5,7,140,142 in the stimulatory effects of other hormones and cytokines, such as 1,25-dihydroxyvitamin D₃, interleukin-1, and interleukin-11. In addition, Collins and Chambers⁴¹ found that prostaglandins can be substituted for 1,25-dihydroxyvitamin D₃ as a differentiating factor in cultures of bone-marrow stromal cells. In contrast to the stimulatory effect of prostaglandins on the formation of osteoclasts in cultures of murine bone-marrow and spleen cells, prostaglandin E₂ was found by Quinn et al.¹²¹ to inhibit profoundly the formation of osteoclasts in cultures of UMR 106 cells and monocytes; however, when monocytes were cultured with ST2 cells, these investigators found that prostaglandin E₂ stimulated the formation of osteoclasts¹²⁵. These findings indicate that the effect of prostaglandins on the formation of osteoclasts from monocyte precursors is highly dependent on the type of stromal cell supporting the differentiation of osteoclasts.

Evidence has already been presented that 1,25-dihydroxyvitamin D₃ is essential for the formation of osteoclasts, particularly in the differentiation of osteoclast precursors. It has been reported⁸⁶ that this activity requires the presence of osteoblasts, which, unlike osteoclasts, possess cytosolic receptors for 1,25-dihydroxyvitamin D₃; 1,25-dihydroxyvitamin D₃ has been localized to the nucleus of osteoblasts, which suggests that this hormone acts through a nuclear mechanism characteristic of other steroid hormones¹⁶⁶. Kurihara et al.⁸⁸ showed that 1,25-dihydroxyvitamin D₃ can act on highly purified populations of precursors for osteoclastic multinucleated cells in cultures of human bone marrow; this indicates that these osteoclast precursors, unlike mature osteoclasts, possess receptors for 1,25-dihydroxyvitamin D₃. It should be noted that monocytes and macrophages possess receptors for 1,25-dihydroxyvitamin D₃ and that 1,25-dihydroxyvitamin D₃ can also be a secretory product of these cells^59,107. 1,25-dihydroxyvitamin D₃ induces differentiation of murine and human myeloid and monoblastic leukemia cells along the monocyte pathway; it also enhances their adhesion to substrates, production of monocyte-specific enzymes, and expression of mononuclear phagocyte-associated antigens, as well as their capacity to bind and degrade devitalized bone particles^23,92,157. 1,25-dihydroxyvitamin D₃ also promotes the fusion of monocytes and macrophages to form macrophage polykaryons^1,49. Although these cells have not been shown to express characteristics of osteoclasts, this is to be expected, as these characteristics have been sought in the absence of osteoblasts or other stromal cells that primarily mediate the differentiation-stimulating effect of 1,25-dihydroxyvitamin D₃ on osteoclast precursors.

It appears that these bone-resorbing agents act through osteoblastic and bone-marrow stromal cells to influence the formation of osteoclasts (Fig. 1). They interact with distinct cell-surface receptors and have distinct transduction pathways in these cells. Cell-to-cell contact between osteoclast precursors and either osteoblasts or stromal cells is also necessary for the generation of osteoclasts, and it has been proposed that these various agents act through a final common pathway to induce the production of a critical common membrane factor on osteoblasts or specific stromal cells. This hypothetical factor, which has been called osteoclast differentiation factor or stromal osteoclast-forming activity^142,143, then recognizes osteoclast progenitors that have been primed by exposure to hematopoietic growth factors, such as macrophage colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and interleukin-3.

The importance of osteoblasts and other stromal cells in the formation of osteoclasts has already been alluded to. These cells are also known to be similarly crucial for the initiation and hormonal regulation of osteoclastic bone resorption. Rodan and Martin¹²⁸ originally proposed the hypothesis that the cells of the osteoblastic lineage that line the bone surface control osteoclastic bone resorption by influencing the formation and function of osteoclasts. The cells of the osteoblastic lineage that line the bone surface are classified as either osteoblasts or bone-lining cells. The former are plump, cuboidal, or polygonal cells that contain an abundance of alkaline phosphatase; they have a large amount of rough endoplasmic reticulum and Golgi apparatus, which corresponds to their location at sites where osteoid and new bone is being laid down. Bone-lining cells are flat and elongated, and they cover most of the bone surface in the adult skeleton; they have few organelles and are thought to represent either resting or relatively inactive osteoblasts or a population of bone cells that may undergo transformation to active osteoblasts. It has been proposed that one means by which cells of the osteoblastic lineage control osteoclastic bone resorption is by the cellular retraction of these cells covering the bone surface⁷⁶. In this way, the bone matrix beneath these cells is then exposed to the action of osteoclasts. Another means whereby osteoblasts may initiate osteoclastic bone resorption is by their digestion of the unmineralized organic matrix that is believed to cover all bone surfaces. Osteoclasts are known to avidly resorb bone in which the unmineralized layer of organic material has been removed from the surface, whereas their resorptive activity is terminated when the mineralized component of the bone matrix is removed^35,36. Osteoblasts are known to produce collagenase and tissue plasminogen activator in vitro, both of which are enzymes that can degrade the organic matrix of bone; increased amounts of these neutral proteases and decreased amounts of protease inhibitors are synthesized in the presence of parathyroid hormone and other bone-resorbing hormones^64,69,133. Synthesis of collagenase is increased and collagenase has also been identified in bone-lining cells in situ in pathological conditions involving increased resorption of bone^42,132. These findings suggest a role for protease-secreting cells as accessory cells in resorption of bone. Osteoblast collagenase, produced as a procollagenase and activated possibly by a plasminogen activator and plasmin proteolytic cascade, under the actions of bone-resorbing agents, has been proposed to be necessary for the removal of the unmineralized osteoid that covers the bone surface; osteoclasts then have access to mineralized matrix, which is a stimulus for the initiation and activation of osteoclastic resorption of bone¹⁵⁹. Other cells that release neutral proteases, such as macrophages and tumor cells, may similarly influence this process by releasing neutral proteases that degrade the organic matrix and, thus, independently activating osteoclastic resorption of bone.

Osteoblasts are known to be the primary cell targets for many local and systemic hormonal factors that influence osteoclastic resorption of bone. This is evidenced by the fact that the bone cells that possess receptors for the major systemic bone-resorbing hormones (parathyroid hormone and 1,25-dihydroxyvitamin D₃) are osteoblasts and not osteoclasts^86,131; it is also apparent from the effect that these hormones have on the resorptive activity of osteoclasts, as observed in the scanning electron microscopy bone-resorption assay. These findings suggest that parathyroid hormone and 1,25-dihydroxyvitamin D₃ stimulate osteoclastic lacunar bone resorption only when osteoblasts are added to the culture system^38,93,94; these hormones have no effect on lacunar bone resorption when they are added to cultures of osteoclasts alone. Similarly, local factors that are known to stimulate osteoclastic resorptive activity, such as prostaglandins, cytokines, and growth factors, do not act directly on osteoclasts but rather through osteoblasts, which mediate the hormonal stimulation of osteoclastic resorptive activity^38,154,155. The nature of this osteoblastic signal that stimulates osteoclastic resorptive activity is not known. It has been postulated that, under the influence of bone-resorbing hormones, osteoblasts release a soluble factor or second messenger that directly stimulates osteoclastic resorption of bone^154,155 or, alternatively, that osteoblasts elaborate a matrix or membrane-bound factor that stimulates resorptive activity after contact with mature osteoclasts⁵³. In contrast, the major inhibitors of osteoclastic resorptive activity, calcitonin and prostaglandins, act directly on osteoclasts^34,36. The inhibitory effect of these hormones can be overcome by adding osteoblasts to these cultures³¹. It should be recognized that the stimulatory effects of hormones on the resorptive activity of osteoclasts are often difficult to separate from those that influence the formation of osteoclasts⁹⁸.

Cellular and Hormonal Mechanisms Associated with Pathological Bone Resorption

Osteoclasts are highly specialized for the function of bone resorption, and it is not surprising that they are commonly found at the site of osteolysis associated with systemic bone diseases, such as hyperparathyroidism and Paget disease, and localized osteolytic lesions, such as primary or secondary osteolytic tumors or the membrane around loose joint implants. In these conditions, the resorption of bone results from an increase in the number or activity of osteoclasts, or both, and can be regarded as a consequence of a disturbance in the mechanisms that govern the formation, activation, and function of these cells.

Knowledge of the cellular and hormonal mechanisms governing the formation and activity of osteoclasts provides important information on the pathogenesis of metabolic bone diseases, in which generalized osteolysis is commonly a feature. Thus, in hyperparathyroidism, the increase in bone resorption may be the consequence of several known effects of parathyroid hormone on the biology of the osteoclast; these effects include an increase in the number of osteoclasts as a result of the stimulation, by parathyroid hormone, of the formation of osteoclast precursors from bone-marrow progenitors and of osteoblast-mediated differentiation of osteoclast precursors to functional osteoclasts. These effects may be a direct result of the increased levels of parathyroid hormone in the serum or may be mediated indirectly by promotion of the renal 1-hydroxylation reaction that leads to the formation of 1,25-dihydroxyvitamin D₃, which is an essential cofactor in the differentiation of osteoclasts. The number of osteoclasts increases rapidly (within hours) after the administration of parathyroid hormone. This also suggests that a resident population of osteoclast precursors is normally present in bone and that parathyroid hormone and other hormones stimulate the formation of mature osteoclasts from this primed cell population²⁵. Parathyroid hormone may also initiate and activate osteoclastic resorption of bone by stimulating the release of neutral proteases by osteoblasts or by inducing a morphological change in the shape of these cells; either mechanism provides a means whereby osteoclasts have access to the underlying mineralized matrix and can begin resorption of bone. Parathyroid hormone and other systemic osteotropic hormones may also influence osteoclastic resorption by regulating the production of cytokines^93,143. This may have an effect on both the formation and the activity of osteoclasts. The synthesis of interleukin-6 and interleukin-11, both products of stromal cells and osteoblasts, is promoted by parathyroid hormone, 1,25-dihydroxyvitamin D₃, and parathyroid hormone-related peptide. Interleukin-6 and interleukin-11 are now thought to be important in the formation of osteoclasts that is stimulated by parathyroid hormone and 1,25-dihydroxyvitamin D₃.

Interleukin-6 appears to play a central role in the pathogenesis of osteoporosis associated with the loss of gonadal function. The production of interleukin-6 by cultured bone-marrow stromal cells and osteoblastic cell lines is inhibited by estrogens⁵⁷, and estrogens also inhibit the transcriptional activity of the human interleukin-6 gene promoter^119,126. In studies of mice from which the gonads had been removed, the formation of osteoclasts was enhanced through an increase in the production of interleukin-6 in the microenvironment of the bone marrow⁷⁵. The number of colony-forming units for granulocytes and macrophages and the number of osteoclasts also were increased in short-term cultures of bone-marrow cells from mice from which the ovaries had been removed. In addition, when 17-estradiol or a neutralizing antibody against interleukin-6 was administered to these mice, the number of osteoclast progenitors did not increase⁵⁷. There appears to be a strong association between a deficiency of gonadal hormones and the production of interleukin-6 in hematopoietic tissues; this results not only in the stimulation of the formation of osteoclast precursors but also in the cell division of early progenitors and the differentiation of other hematopoietic cell lines. Interleukin-1 and tumor necrosis factor have also been reported to be involved in the pathogenesis of postmenopausal osteoporosis^81,83. Ovariectomy in mice was found to increase the secretion of interleukin-1 and tumor necrosis factor (but not interleukin-6) by bone-marrow cells as well as the formation of osteoclast-like multinucleated cells positive for tartrate-resistant acid phosphatase in cultures of bone-marrow cells treated with 1,25-dihydroxyvitamin D₃. This increase in the formation of multinucleated cells induced by ovariectomy was prevented by treatment with an antagonist to the interleukin-1 receptor and tumor necrosis factor binding protein, as well as estrogens. These factors also decreased resorption of bone in vivo and in vitro.In vivo studies of rats have also shown that treatment with an antagonist to the interleukin-1 receptor decreases bone loss and resorption of bone after ovariectomy. These data suggest that interleukin-1 or mediators induced by interleukin-1 may have an important role in the bone loss associated with a deficiency of estrogen.

Interleukin-6 has also been reported to be involved in the pathogenesis of bone resorption associated with Paget disease^129,130. Osteoclast-like multinucleated cells readily form in long-term cultures of human bone-marrow cells from patients who have Paget disease⁸⁷; these osteoclast-like cells form more quickly, have increased levels of tartrate-resistant acid phosphatase, and are much more responsive to 1,25-dihydroxyvitamin D₃, compared with cells formed in cultures of normal human bone cells. High levels of interleukin-6 have been found in the conditioned medium of the cultures of pagetic bone-marrow cells, and this conditioned medium has been found to promote the formation of osteoclasts in cultures of normal human bone-marrow cells^129,130. The bone-marrow plasma and peripheral blood from patients who have Paget disease also contain elevated levels of interleukin-6. Osteoclasts from patients who have Paget disease also express mRNA for interleukin-6, the receptor for interleukin-6, and the nuclear transcription factor for interleukin-6; this is in contrast to normal osteoclasts, which have been shown⁷⁴ to express only mRNA for interleukin-6. Thus, both the increased production of interleukin-6 and the response to this cytokine may lead to an autocrine amplification of the stimulation of the formation and activity of osteoclasts by interleukin-6. Stromal cells that have been aspirated from the bone marrow of patients who have Paget disease have also been found to enhance the growth of osteoclast precursors isolated from both pagetic and normal bone marrow^43,129. This suggests that stromal cells from the microenvironment of the bone marrow are the main source of interleukin-6 and that they are involved in the increased formation of osteoclasts that is associated with Paget disease.

The level of interleukin-6 has also been increased in the serum of some patients who have multiple myeloma^84,129. Both in vivo and in vitro models that have been used to study the formation of osteoclasts in association with multiple myeloma have also shown that the osteolysis associated with this condition depends on interleukin-6. Interleukin-6 is not produced by myeloma cells but, as in Paget disease, it is probably produced by bone-marrow stromal cells in patients who have multiple myeloma. Interleukin-6 is a known growth factor for myeloma cells, and treatment with anti-interleukin-6 antibodies has been found to decrease the tumor burden in some patients who have multiple myeloma¹²⁹. Myeloma cells also produce numerous locally acting cytokines and growth factors that can stimulate the formation and activity of osteoclasts; these include interleukin-1, tumor necrosis factor-alpha, and lymphotoxin (tumor necrosis factor-ß). Interleukin-6 has also been found to promote the resorption of bone that is associated with rheumatoid arthritis and vanishing bone disease^44,85.

The giant-cell tumor of bone is an uncommon osteolytic primary bone tumor that is characterized by the presence of abundant multinucleated osteoclast-like giant cells and a distinct mononuclear cell component. The osteoclastic nature of the giant cells in giant-cell tumor of bone has long been suspected from their close morphological and cytochemical resemblance to osteoclasts⁶¹. The giant cells also fulfill all of the essential criteria for defining osteoclasts: they possess abundant calcitonin receptors¹¹⁰, respond to calcitonin with a rise in cyclic adenosine monophosphate⁵⁸, and are capable of forming resorption pits on bone slices in a manner identical to that of osteoclasts³⁷. In fact, the now commonly used technique of cell culture on bone slices to determine evidence of lacunar resorption was first carried out with use of cells isolated from a giant-cell tumor of bone¹⁶. Ruffled borders and clear zones, ultrastructural features that are characteristic of the osteoclast, have been seen on these giant cells forming these resorption lacunae⁸². This is in contrast to the somewhat poor development of these features in giant cells in situ⁶¹. The giant cells of giant-cell tumor of bone are also positive for tartrate-resistant acid phosphatase⁶² and have an antigenic phenotype identical to that of osteoclasts^15,73, expressing the same restricted range of macrophage-associated antigens; this pattern of expression is particularly useful in distinguishing the giant cells of giant-cell tumor of bone from giant cells in other giant-cell-rich tumors and tumorlike lesions of bone, such as non-ossifying fibroma and aneurysmal bone cyst⁴⁶. The only other tumor in which giant cells have been reported to show an identical osteoclast-like phenotype is giant-cell granuloma of the jaw⁵².

Localized abnormal resorption of bone may result from a variety of causes, but it is most often associated with neoplastic and inflammatory lesions in bone. Tumor cells and inflammatory cells release numerous cytokines, prostaglandins, and other local factors that enhance the bone-resorptive activity of mature osteoclasts^45,102,105; this effect is mediated indirectly by osteoblasts. Another means whereby these cells can contribute to osteoclastic bone resorption may be by release of proteases that degrade the organic matrix that covers bone surfaces; this could lead to exposure of the mineralized matrix, which activates osteoclastic bone resorption^35,36. The release of prostaglandins, cytokines, and growth factors by inflammatory and tumor cells may also act on osteoblasts and stromal cells to regulate the formation of osteoclasts from osteoclast precursors.

Macrophages are a major component of the host cellular response to neoplastic and inflammatory lesions in bone^27,162. Stimulated by cytokines found in inflammatory lesions, tumor cells and osteoblastic cells produce chemoattractant proteins that induce the recruitment of monocytes into these lesions^167,170. As noted earlier, it has been shown that, among the macrophage population of various extraskeletal normal and abnormal (neoplastic and inflammatory) tissues, there are cells of macrophage phenotype that are capable of differentiation into functionally mature osteoclasts. Tumor-associated macrophages derived from primary carcinomas of the lung in humans and of the breast in mice^12,120,124, as well as inflammatory foreign-body macrophages derived from granulomas induced by the wear particles of implanted biomaterials^117,123, have all been shown to be capable of osteoclastic differentiation. These macrophages require the same cellular and hormonal conditions for osteoclastic differentiation as those of osteoclast precursors derived from hematopoietic tissues (the presence of osteoblasts or other specific stromal cells and 1,25-dihydroxyvitamin D₃).

As indicated earlier, tissue macrophages are not a homogeneous cell population; rather, they are heterogeneous in terms of their morphology, function, immunophenotype, and enzyme histochemistry⁵⁹. This heterogeneity is also reflected in their proliferative potential; approximately 5 per cent of tissue macrophages are capable of further division¹⁶¹. As not all tissue macrophages appear to undergo osteoclastic differentiation in vitro, this subpopulation of relatively immature macrophages may represent that fraction that undergoes osteoclastic differentiation when these cells are placed in the cellular and hormonal tissue microenvironment of bone. A corollary of this phenomenon would be that more such immature cells capable of osteoclastic differentiation would be found in tissues containing numerous macrophages. As many osteolytic lesions of bone are associated with the presence of a heavy infiltrate of macrophages, this mechanism of macrophage-osteoclast differentiation could represent one means whereby abnormal resorption of bone is effected. Much of the work on macrophage-osteoclast differentiation has been carried out with populations of murine mononuclear phagocytes, but human tumor-associated macrophages have been shown to be capable of osteoclastic differentiation¹²; there is also evidence that human monocytes, peritoneal macrophages, and macrophages derived from the membrane surrounding a loose prosthesis, from carcinomas of the breast, and from the synovial membrane of a joint affected by rheumatoid arthritis can differentiate into mature osteoclasts capable of extensive lacunar bone resorption (unpublished observation).

This mechanism of macrophage-osteoclast differentiation is probably of particular relevance to lesions with which there is rapid destruction of bone associated with a heavy infiltrate of macrophages, such as in the form of aseptic loosening termed aggressive granulomatosis; rapid extensive osteolysis is seen in association with a pronounced foreign-body macrophage response to the formation of numerous wear particles from implanted biomaterials^65,134. Inflammatory granulomas are known to contain an increased number of such phagocytes with proliferative potential¹⁴¹, and within the heavy infiltrate of macrophages that is seen with conditions such as aggressive granulomatosis there is probably an increased number of such relatively immature macrophages capable of undergoing osteoclastic differentiation. Macrophages responding to particles of biomaterials that are commonly used for arthroplasty have all been shown to be capable of this form of osteoclastic differentiation¹¹⁷; therefore, the macrophage-osteoclast differentiation may also contribute to the more common slowly progressive osteolysis of aseptic loosening by this mechanism. This would operate in addition to other known mechanisms of promotion of bone resorption by macrophages, such as the release of humoral factors that stimulate resorption by resident osteoclasts. Other lesions characterized by a heavy infiltrate of macrophages (such as those associated with Gaucher disease or histiocytosis X) may similarly generate osteoclast-like bone-resorbing cells from within this infiltrate.

Scanning electron microscopic studies of the bone resorption by macrophages associated with extraskeletal inflammatory and neoplastic lesions have also shown that, when these cells are incubated alone (without stromal cells) on bone slices, they appear to be independently capable of a type of low-grade bone resorption, characterized by roughening of the bone surface with exposure of mineralized collagen fibers. This type of resorption is easily distinguished morphologically from that caused by osteoclasts, which results in the formation of well defined excavations of measurable depth in bone. This kind of low-grade resorption was first noted in association with macrophages and macrophage polykaryons isolated from extraskeletal inflammatory and neoplastic tissues containing many of these cells, such as the pseudocapsule of loose prosthetic components and giant-cell tumors of the tendon sheath^13,17. It has also been observed when inflammatory and tumor-associated macrophages have been cultured on bone slices, both in the presence and in the absence of bone-marrow stromal cells^12,120,123. Hypothetically, this type of resorption could be carried out by mononuclear phagocytes that have not been exposed to or are not responsive to the cellular and hormonal signals necessary for osteoclastic differentiation. Many previous studies have provided indirect evidence of degradation of the bone mineral and organic matrix by macrophages and their fused products, macrophage polykaryons^49,104,152. Extensive low-grade surface resorption may be the functional and morphological correlate of this phenomenon. The collective effect of such low-grade resorption by large numbers of mononuclear phagocytes could be quantitatively substantial if effected over a long period of time. This type of bone resorption could partially account for the osteolysis associated with some slowly progressive inflammatory and neoplastic lesions of bone, such as the gradual enlargement of established osteolytic bone metastases, which occurs in the apparent absence of osteoclasts⁵⁵. This could result from the action of tumor-associated macrophages or be a function of tumor cells themselves; the latter have also been shown to be capable of releasing mineral and organic matrix components from devitalized bone particles⁴⁸. Tumor cells, however, are not capable of lacunar bone resorption, even under conditions in which tumor-associated macrophages have been shown to be capable of differentiation into osteoclastic bone-resorbing cells¹²⁴.

In summary, recent studies have confirmed that the osteoclast is a member of the mononuclear phagocyte system; it is derived from the pluripotential hematopoietic stem cell, follows the monocyte-macrophage hematopoietic lineage, and has a precursor that circulates in the monocyte fraction. In the cellular and hormonal microenvironment of bone, monocytes, macrophages, and their less mature hematopoietic precursors have all been shown to be capable of differentiating into lacunar bone-resorbing osteoclasts positive for calcitonin receptors. Osteoclastic differentiation requires the presence of bone-marrow stromal cells and osteoblasts, and these cells also have a central role in the activation and hormonal control of osteoclastic resorption of bone. In conditions characterized by either generalized or localized abnormal resorption of bone, the complex cellular and hormonal interactions governing the formation and function of osteoclasts have been found to be abnormal, leading to an increase in the number or activity of osteoclastic bone-resorbing cells; other cells, such as macrophages and tumor cells, may independently contribute to the degradation of bone matrix components, but the sole cellular agent of lacunar bone resorption is the mature osteoclast.

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