Osteal macrophages: A new twist on coupling during bone dynamics

Bone 43 (2008) 976–982

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Bone j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b o n e

Review

Osteal macrophages: A new twist on coupling during bone dynamics Allison R. Pettit a,⁎, Ming K. Chang a, David A. Hume b, Liza-Jane Raggatt a,⁎ a b

The University of Queensland, Institute for Molecular Bioscience, Cooperative Research Centre for Chronic Inflammatory Diseases, Brisbane, QLD 4072, Australia The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9PS, Scotland, UK

a r t i c l e

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Article history: Received 9 July 2008 Revised 18 August 2008 Accepted 21 August 2008 Available online 16 September 2008 Edited by: T.J. Martin Keywords: Macrophage Osteoblast Osteoimmunology Bone modelling Bone remodelling

a b s t r a c t Osteoimmunological interactions are central to maintaining bone homeostasis and are key mechanisms in bone pathology. Macrophages are highly adaptable cells with pleiotropic actions. They have important roles in development, homeostasis and both innate and adaptive immunity. Macrophages can have broad ranging effects on bone, particularly in pathologic situations, but they are most commonly considered for their in vitro potential as an osteoclast precursor. We have recently shown that, like most tissues, the endosteum and periosteum contain a population of resident tissue macrophages (OsteoMacs) that impact on the bone formation process and are likely to play important roles in the bone niche. This review discusses the wider impact of macrophages in bone homeostasis and disease and proposes novel roles for OsteoMacs in bone modelling and remodelling. © 2008 Elsevier Inc. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . Macrophages . . . . . . . . . . . . . . . . . . . . . . . . . . Macrophages participate in bone pathology, repair and calcification Osteal tissues contain a resident macrophage population . . . . . Are OsteoMacs osteoclast precursors? . . . . . . . . . . . . . . OsteoMacs direct osteoblast function/mineralization. . . . . . . . OsteoMacs participate in bone modelling . . . . . . . . . . . . . Do OsteoMacs contribute to bone remodelling? . . . . . . . . . . Conclusions and future directions . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Introduction The developing understanding of the intimate relationship between the immune system and bone biology has culminated in the emerging field of osteoimmunology. During homeostasis there is a bi-directional trophic relationship between bone and the immune system but pathology often results in destructive ramifications for bone, as is the case in rheumatoid arthritis [1]. In pathological conditions, macrophages can directly and indirectly contribute to both anabolic and catabolic bone outcomes. However macrophage participation in physiologic bone turnover has not been explored. Given the diverse functional abilities of macrophages and their relationship to osteoclasts, as well as their broad-spectrum involvement in ⁎ Corresponding authors. Fax: +61 7 3346 2101. E-mail addresses: [email protected] (A.R. Pettit), [email protected] (L.-J. Raggatt). 8756-3282/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2008.08.128

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development, tissue homeostasis and immunity, we became intrigued by the potential osteoimmunological contributions of these cells. Macrophages Macrophages are heterogeneous mononuclear cells derived from hematopoietic stem cells along the myeloid lineage (Fig. 1) [2]. The survival, proliferation and differentiation of myeloid lineage cells, including macrophages, is dependent on the cytokine CSF-1 [3]. Infection, inflammation and tissue injury results in rapid recruitment of monocytes from peripheral blood to the ‘insult’ site (Fig. 1). These monocytes then differentiate into immune macrophages that drive innate and adaptive immune responses [4]. Under physiological conditions, monocytes migrate into virtually all tissues of the body and differentiate into resident tissue macrophages (Fig. 1). While less renowned than their inflammatory/infection counterparts, resident

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Fig. 1. Schematic of macrophage and osteoclast development. Macrophages and osteoclasts develop from hematopoietic stem cells and subsequently myeloid precursor cells within bone marrow. Colony stimulating factor (CSF)-1 binds to the CSF-1 receptor (CSF-1R) on myeloid precursors and drives their proliferation and differentiation into peripheral blood monocytes or osteoclast (OC) precursors [2,85]. Under physiologic conditions monocytes are recruited into virtually all tissues (‘normal tissues’) and subsequently undergo tissue specific differentiation into resident tissue macrophages. Monocytes are also actively recruited to sites of infection, inflammation or tissue injury and rapidly differentiate into inflammatory macrophages and secrete a wide variety of cytokines and growth factors that are polarized depending on the stimulus [2,4]. Most murine resident tissue and immune macrophages can be identified by expression of the F4/80 antigen [4]. Osteoclast precursors circulate in blood (also located in bone marrow, not shown on figure) and express receptor activator of NFκB (RANK). Binding of RANK ligand to this receptor is the most potent pro-osteoclastogenic signal. Expression of osteoclast-associated proteins tartrate resistant acid phosphatase (TRAP) and calcitonin receptor (CTR) are induced during the differentiation and maturation/activation stages, respectively [9]. There is plasticity within the myeloid lineage and most cells, including inflammatory macrophages (broken arrow) [40,41], can be differentiated into osteoclasts in vitro.

tissue macrophages play important global and tissue specific roles in developmental and ongoing homeostatic processes [2,4]. For example: microglia, tissue macrophages in the brain, are responsible for removal of redundant or inappropriate neuronal processes during brain development [5] and Kupffer cells, tissue macrophages of the liver, participate in liver function by removing particulate matter from portal circulation and eliminating/detoxifying gastro-intestinally absorbed microorganisms/toxins [6]. Tissue specific adaptation results in heterogeneity between resident macrophage subpopulations [4,7,8], between different tissues (e.g. alveolar macrophages in the lung, microglial cells in the central nervous system and Langerhans' cells in the epidermis [4]) and within tissues (e.g. splenic metallophilic, white pulp, marginal zone and red pulp macrophages [4]). Macrophages are also closely related to bone resorbing osteoclasts, but can be distinguished from these cells through morphological features and the expression of differential marker proteins (Fig. 1) [4,9]. Resident tissue macrophages share many functional properties. They are an integral component of the innate and adaptive immune system and continuously survey and respond to changes in their local environment including apoptotic cells, pathogens and cellular/ matrix debris [10]. Tissue macrophages also have the capacity to secrete a wide range of regulatory molecules in response to environmental stimuli [11] and often have tissue specific trophic roles [7,12]. The F4/80 monoclonal antibody [13] has been widely used to identify macrophages in murine tissues. Most resident tissue macrophages express this surface molecule (exceptions being alveolar macrophages and some splenic macrophages [8]) and as F4/80 is rapidly down regulated during osteoclastogenesis [14], it is useful in distinguishing mature macrophages from osteoclasts and their immediate precursors (Fig. 1).

Macrophages participate in bone pathology, repair and calcification Macrophages are known to influence aspects of bone biology and pathology. They are present during multiple stages of fracture repair, produce mesenchymal growth factors at the facture site [15] and have been associated with more stable callus formation [16]. Macrophages have also been implicated in arteriole calcification [17]. Co-culture of macrophages with osteoblast-like calcifying vascular cells (CVC) [18] or human vascular smooth muscle cells [19] resulted in increased alkaline phosphatase expression and matrix mineralization by these cells. Production of tumour necrosis factor [18,19] and oncostatin M [19] has been implicated as the molecular mechanism by which macrophages drive vascular calcification. Depletion of macrophages in osteoarthritic models dramatically reduced osteophyte formation [20–22] suggesting macrophages play a critical role in ectopic bone deposition. Additionally, macrophages are associated with sites of pathologic bone loss [23,24] and also contribute to systemic pathologic mechanisms in an animal model of osteoporosis [25]. These observations link macrophages to ectopic mineralization, both extremes of bone disease and bone repair. Macrophages have the potential to contribute to bone biology via expression and secretion of osteo-active cytokines, matrix metalloproteinases (MMP) and bone morphogenetic proteins (BMP) [21,23,26]. Older data implicating macrophages as the cells responsible for mediating the reversal phase of bone remodelling are also relevant [27]. The presence of F4/80+ macrophage-like cells on osteal surfaces was reported 24 years ago [28] but the concept that macrophages occupy a defined niche in osteal tissues had not been

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further developed. It is clear that macrophages may participate in bone homeostasis and disease. However detailed description of a resident macrophage population in osteal tissues and direct evidence supporting bone-relevant functional potential of macrophages, particularly during homeostasis, has been lacking. Osteal tissues contain a resident macrophage population We have recently extended the original observation that macrophages are associated with bone surfaces [28] by clearly establishing that periosteal and endosteal tissues contain a discrete population of resident tissue macrophages that we have named OsteoMacs (in line with traditional bone cell nomenclature) [29]. OsteoMacs constitute approximately one sixth of the total cells within osteal tissues [29]. They achieve extensive coverage of bone surfaces through their stellate morphology ([29] and Figs. 2A, B and C), suggesting the formation of a comprehensive OsteoMac network covering the entire bone surface. We have shown CD68+TRAPneg macrophage-like cells are located on human osteoarthritic bone surfaces [29] and have observed that a subset of cells grown from human periodontal bone explants cultures are CD68+ (unpublished data), indicating that OsteoMacs are also a component of human osteal tissues. It is clear that, like most other tissues, osteal tissues contain a resident population of macrophages. As is the case for other tissue macrophage populations [4,7,8], we predict that OsteoMacs will have a subtly different and unique phenotype and transcriptome that is dictated by

their tissue microenvironment. Determination of OsteoMac unique characteristics will be greatly aided by successful purification of this cell population and will determine if OsteoMacs can be specifically targeted for depletion or transgenic strategies. We propose that OsteoMacs perform both ‘global’ and tissue specific functions within the bone microenvironment and through these functions, contribute to both bone physiology and pathology. A ‘global’ tissue macrophage function that OsteoMacs are likely to perform is immune surveillance in the bone microenvironment. Previous studies have reported macrophage-related functions such as phagocytosis [30,31], detection of bacterial products [32,33] and antigen presentation [31,34] in primary osteoblast cultures. Given our recent demonstration that murine osteoblast preparations co-isolate OsteoMacs [29], we argue that these observed functions need to be more definitively assigned. We provide evidence that it is the OsteoMacs within primary murine osteoblast cultures that detect and respond to patho-physiologic levels of lipopolysaccharide (LPS) [29]. Some studies using human osteoblast-like cells to demonstrate classical macrophage-like functionality within these cultures [30,31,34,35] have attempted to substantiate that they are using homogeneous osteoblast cells. However, macrophage expression of the osteoblast marker proteins used (primarily alkaline phosphatase and osteocalcin) has been reported under certain conditions [36,37]. Co-expression of CD68 and alkaline phosphatase has been reported in cultures of human peripheral blood monocytes stimulated with IL-4 and in a monocyte-derived in vitro granuloma model [36].

Fig. 2. Osteal tissues contain a population of resident tissue macrophages (OsteoMacs). Immunofluorescence and immunohistochemistry were used to characterise the presence and distribution of macrophages within osteal surface tissues. (A and B) En face views of the endosteum of a bone fragment, harvested from 8 week old MacGreen transgenic mice (in which the csf1r promoter drives the expression of enhanced green fluorescent protein (eGFP) resulting in ‘green’ macrophages and other myeloid cells) and cultured for 1–2 weeks in the absence of exogenous CSF-1. Transmitted (A) and epi-fluorescence (B) microscopy of the same bone fragment demonstrating numerous eGFP+ cells over the entire endosteal surface. Bone pieces were visualized using an Olympus IX-70 epi-fluorescence light microscope original magnification x10. (C) Immunohistochemical staining using rat anti-mouse F4/80 antibody (AbD Serotec, Kidlington, Oxford) on a mouse long bone section showing a planar cut through the periosteal bone surface. (D) Rat IgG2b isotype control (AbD Serotec) staining on a serial bone section. Arrowhead in C and D shows a multinucleated osteoclast-like cell that is juxtaposed to a small fragment of bone (b) that is visible in the section plane. Sections were deparaffinized and rehydrated followed by antigen retrieval by enzymatic digestion using 0.37% trypsin. Sections were incubated for 60 min in serum block (10% FCS plus 10% normal goat serum in TBS) and then incubated with the primary antibody for 60 min. Endogenous peroxidase activity was blocked using 3% H2O2. Sections were subsequently treated for 30 min with a biotinylated F(ab′)2 fragment of goat anti-rat immunoglobulin (Santa Cruz, California, USA), followed by horseradish peroxidase-conjugated streptavidin (DakoCytomation, Glostrup, Denmark) and developed using diaminobenzidine chromogen (DakoCytomation). Sections were counterstained with hematoxylin and mounted using permanent mounting media, original magnification ×20.

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Additionally, ex vivo [36] and in situ [37] observations similarly revealed co-expression of CD68, alkaline phosphatase and osteocalcin in macrophage cells associated with failed prostheses. Observations reporting potential cross-lineage plasticity of osteoblast and hematopoietic cells in vitro [36,38,39] adds further difficulty to clearly assigning the cell responsible for any specific function reported in primary ‘osteoblast’ cultures. Our description of a more complex cellular composition of osteal tissues and that cell preparations generated from this tissue recapitulate this, confirm the need for primary osteoblast cultures to be more stringently purified when undertaking functional studies.

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Are OsteoMacs osteoclast precursors? Based on lineage relationship [14] and anatomical location [29], an immediate and obvious assumption to make is that the primary role of OsteoMacs is to serve as in vivo osteoclast precursors. OsteoMacs are clearly not osteoclasts, as osteoclasts do not express the F4/80 antigen [13,14] and F4/80+ OsteoMacs in situ are not multinucleated (Fig. 2 and [29]). Bone marrow-derived macrophages (BMM) [40] and rheumatoid synovium-derived macrophages [41] can be differentiated into osteoclasts in vitro, supporting that mature macrophages can serve as osteoclasts precursors. However, there are alternative, more primitive

Fig. 3. Proposed roles for OsteoMacs in bone modelling and remodelling. (A) Bone Modelling i) Activation phase: OsteoMacs are intercalated with bone lining cells on resting bone surfaces. Both of these cells detect and respond to anabolic signals within the bone microenvironment. ii) Bone formation phase: OsteoMacs proliferate at, or are recruited to, the modelling site (likely through increased local CSF-1 production by bone lining cells/immature osteoblasts). Concomitantly OsteoMacs mediate the proliferation and differentiation of osteoblast at the formation site and subsequently form the canopy structure over these osteoblasts. OsteoMacs within the canopy provide signals that promote osteoblast-mediated matrix formation and ultimately mineralization. iii) Termination phase: following completion of bone formation, OsteoMacs and bone lining cells return to a resting state. The ‘termination signal’ is not yet well characterised and may be mediated through expression of an anti-anabolic factor(s) (candidate molecules include sclerostin [86] and noggin [87]) or removal of anabolic factor(s). (B) Bone Remodelling i) Activation and Resorption phase: Given the sentinel capacity of macrophages, we predict that OsteoMacs, in coordination with bone lining cells, participate in the detection of at least some bone remodelling signals, ultimately resulting in osteoblast expression of the pro-osteoclastic molecules RANKL and CSF-1. Pre-osteoclasts are recruited in response to these cytokines and osteoclast formation and resorption follows. As with the bone modelling process, it is anticipated that OsteoMacs undergo proliferation or recruitment to facilitate canopy formation. It is also plausible that OsteoMacs produce signals that regulate both osteoclast formation as well as function. ii) Coupling phase: Osteoclasts provide a ‘coupling signal’ that promotes osteoblast recruitment and the initial stages of bone formation. iii) OsteoMacs are optimally located to communicate with osteoblasts and similarly contribute to the regulation of bone formation. Specifically, OsteoMacs regulate late stage mineralization at the receding end of the basic multicellular unit (BMU), where osteoclasts are no longer present. Bi-directional communication between osteoblasts and OsteoMacs is likely to occur during this process. iv) Termination phase: Bone formation is completed in response to a specific termination signal (as discussed above) or loss of anabolic factors and resting osteal tissue is re-established.

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myeloid cells available within the bone microenvironment and in vitro evidence suggests that these cells can be more efficiently differentiated into osteoclasts [42,43]. This argues against OsteoMacs being the preferred in vivo osteoclast precursor. OsteoMacs, as well as recruited inflammatory macrophages, are more likely to contribute to the osteoclast precursor pool under pathological conditions. Ongoing investigation is required to definitively implicate or eliminate OsteoMacs as the in vivo osteoclast precursor. Alternatively, OsteoMacs activated by local stimuli are likely to produce pro- and anti-osteoclastogenic cytokines such as TNF [44,45], interleukin (IL)-6 [46], IL-1 [44,47] or interferon-β [48,49], and thereby regulate osteoclast generation and function. This hypothesis provides a more direct candidate cellular mechanism to explain why chronic inflammation [50,51] and systemic infection [52,53] often cause osteopenia/osteoporosis. Clearly, systematic investigation of either the direct (acting as a precursor) or indirect (through OsteoMac expression of chemokines/cytokines/growth factors) participation of OsteoMacs in physiologic and pathologic osteoclastogenesis is required. OsteoMacs direct osteoblast function/mineralization Depletion of OsteoMacs from primary murine osteoblast preparations compromised full functional maturation of osteoblasts in classical differentiation cultures [29]. This observation suggested that macrophages have a novel tissue specific function in the bone environment through regulation of osteoblast matrix production and/ or mineral deposition. Induction of osteoclast-specific genes in this culture system was minimal [29], indicating this is not an in vitro recapitulation of the osteoclast–osteoblast coupling that is well documented in vivo [54–56]. Similar to our observations in primary osteoblast cultures, macrophage-specific genes (csf1r, cd14 etc) are also induced in primary mouse chondrocyte differentiation cultures [57]. This raises the possibility that macrophages are also anatomically associated with areas of chondrogenesis and that they potentially act as tropic cells during this process, which is a key step in endochondral ossification. Macrophages can produce many osteo-active factors so their potential to direct the activity of osteoblasts is established. These factors include transforming growth factor β (TGF-β) [58], osteopontin [59], 1,25-dihydroxy-vitamin D3 [60] and BMP-2 [26]. Production of such factors by macrophages promotes myofibroblast activation and extracellular matrix deposition in pathological fibrosis [61,62], and, as discussed above, macrophages enhance vascular calcification in vitro [18,19]. The adaptability of macrophages suggests that their trophic role in bone will be influenced by changes in the microenvironment. We predict that physiologic stimuli promote anabolic function, while inflammatory/infection signals skew OsteoMacs to a catabolic phenotype (i.e. production of pro-osteoclastogenic factors). Indeed, LPS activated macrophages inhibit osteoblast function in vitro [63] and macrophage activation by semisoluble aminated glucan compromises fracture healing [64]. In our hands, in vitro differentiated BMM required elevated extracellular calcium, a characteristic feature of the bone microenvironment, to induce anabolic activity [29]. Overall, OsteoMacs are likely to have a broad range of effects on the bone niche. Developing a clear understanding of anabolic versus catabolic OsteoMac promoting conditions and their subsequent phenotypes will be critical to tapping into the therapeutic potential of this novel biology. OsteoMacs participate in bone modelling Bone modelling is an anabolic process and, unlike bone remodelling, it does not involve balanced coordinated activity of osteoclasts and osteoblasts [56,65,66]. We localised OsteoMacs at bone modelling site (confirmed by absence of osteoclasts in the near vicinity) on cortical diaphyseal endosteal bone surfaces. The OsteoMacs encapsu-

late functionally mature osteoblasts, forming a canopy-like cell structure [29]. The functional importance of OsteoMacs in the bone modelling process was strongly supported by the consequence of macrophage depletion (achieved using the Macrophage Fas-induced apoptosis (Mafia) transgenic mouse model in which conditional macrophage depletion can be induced using synthetic ligand treatment [67]), which disrupted the OsteoMac canopy architecture and resulted in a complete loss of the mature osteoblast bone modelling surface in this anatomical location [29]. At sites of bone remodelling, osteoclasts are thought to provide a ‘coupling signal’ to promote and coordinate osteoblast activity [56]. Given the lineage and functional relationship between macrophages and osteoclast, we propose that OsteoMacs at sites of bone modelling provide a coupling-like signal to osteoblasts. For example TGFβ and ephrin B2 have been implicated as possible coupling factors [56,68] that are made by osteoclast [69,70] and drive bone formation during bone remodelling. Macrophages can produce TGFβ [58] and express ephrin B2 [71] making it possible that OsteoMacs provide these bone forming signals during bone modelling. Our proposed model (Fig. 3) places OsteoMacs within ‘resting’ osteal tissues where they are constantly surveying for alterations in the local environment. In response to anabolic stimuli, they recruit new osteoblast precursors and/or induce the proliferation of adjacent bone lining cells and promote their access/attachment to the bone surface. The OsteoMacs undergo local proliferation, likely driven by osteoblast-derived CSF-1, leading to the formation of the canopy structure. They subsequently provide ongoing anabolic/mineralization signals to the underlying osteoblasts. Once the anabolic stimulus is removed or a specific termination signal has been detected, the OsteoMac cells within the canopy withdraw the anabolic factor(s) and either disperse and/or undergo apoptosis. Subsequently the underlying mature osteoblasts either revert to a bone lining cell phenotype or die and are replaced by resting osteal tissue. Do OsteoMacs contribute to bone remodelling? Bone remodelling does require the sequential and balanced activities of osteoclast-mediated bone resorption and osteoblastmediated bone formation [56,65,72]. Resorption signals are detected by bone lining cells resulting in RANKL and CSF-1 expression, which then promote site-directed osteoclastogenesis and osteoclastmediated bone resorption. It has been proposed that the osteoclast subsequently provides a coupling signal(s) coordinating osteoblast recruitment and differentiation and ultimately quantitatively matched bone deposition and mineralization [56]. The spatial and temporal relationships between these cells are maintained throughout the remodelling process [73] dictating that osteoclasts are only located at the leading edge of the formation phase and have either moved on or undergone apoptosis before formation is completed at any given site [74]. What cellular/molecular mechanism drives osteoblasts to initiate mineralization and complete the remodelling cycle? A cellular canopy structure has been reported over sites of bone remodelling in human bone [75] and was postulated to create an enclosed compartment for local communication and coordination during the complex remodelling process [76]. These canopy cells have been proposed to be bone lining cells based on immunohistochemical staining for alkaline phosphatase, osteocalcin and osteonectin. Under the conditions used in this study, the canopy cells expressed osteocalcin and osteonectin, but the matrix producing cuboidal osteoblasts immediately adjacent to bone surfaces did not [75]. As discussed above, macrophages can express alkaline phosphatase and osteocalcin [36,37] under certain conditions and therefore the study by Hauge et. al. does not rule out the possibility that the canopy cells they described in human bone are macrophages. Visualization of complete basic multicellular units (BMUs) in histological sections of murine bone is rare and determination of

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complex three-dimensional cell structures in the trabecular bone network is difficult, but we have observed that a similar canopy structure encapsulates remodelling sites (including leading resorbing osteoclast, mature osteoblasts and vasculature) in mice and that the canopy cell is an F4/80+ OsteoMac (unpublished data, depicted schematically in Fig. 3B). Osteoclasts may have the dominant role in orchestrating the recruitment, proliferation and initial differentiation of pre-osteoblasts during bone remodelling. But, based on OsteoMac anatomical location, canopy architecture, requirement for maintenance of osteoblast bone modelling surface and their ability to promote optimal osteoblast mineralization in vitro [29], we propose that OsteoMacs are the cell ideally located to perpetuate ‘coupling’ during the remodelling cycle. Specifically, OsteoMacs may direct ongoing matrix deposition and terminal osteoblast mineralization in the tail end of the BMU (Fig. 3B). Acceptance of the central importance of osteoclast–osteoblast coupling in regulating bone dynamics is largely based upon model systems in which osteoclasts are depleted genetically (for Review see [77]). Most of the mutations that affect osteoclasts are likely to also impact on macrophages. However, there are models that provide exceptions to the osteoclast–osteoblast coupling paradigm, providing circumstantial evidence that osteoclasts are not the only cell able to modulate osteoblast function. Osteopetrotic op/op mice lack CSF-1 and have a severe, but not complete, deficiency in both osteoclasts and macrophages [12,78–80]. Compensatory mechanisms have been triggered in this model, such as secondary hyperparathyroidism, so it is likely that this germ-line deficiency does not entirely reflect normal physiology [78]. There is an age related recovery of both osteoclasts and F4/80+ tissue macrophages in these mice that leads to reversal of the osteopetrotic phenotype [12,79]. Interestingly, in op/op mice from approximately 45 days of age, an age at which osteoclastogenesis has been re-established (as evidenced by initiated reversal of the osteopetrotic phenotype), the osteoblast bone formation rate is suppressed by approximately 5 fold compared to littermates [78]. This indicates that in these mice the bone microenvironment does not support normal bone cell dynamics even though osteoclast formation and activity has resumed. Additionally, qualitative analysis of osteoblast morphology and activity in op/op mice suggested that diaphyseal, but not metaphyseal, osteoblast activity is coupled to osteoclast activity after systemic injection of CSF-1 [81]. We would predict that absence of osteoblast/bone lining cell expression of membrane-associated CSF-1 compromises the appropriate reconstitution and distribution of OsteoMacs and that this subsequently contributes to the loss of “coupling” that is evident in op/op mice post osteoclast recovery. It is also clear that the bone formed in op/op mice is abnormal, and in particular that it is poorly mineralized [81,82]. This is pertinent given our demonstration that OsteoMacs are required for optimal osteoblast mineralization in vitro [29]. The deletion of v-ATPase V0 subunit d2 (Atp6v0d2) also results in osteopetrosis due to a failure in osteoclast fusion leading to a marked reduction in osteoclast number in vivo [83]. Atp6v0d2 was identified as an osteoclast-specific gene based on differential expression analysis with macrophages and dendritic cells so this mutation is unlikely to impact on these related cell types [84]. Counter to the osteoclast– osteoblast coupling model, these mice have increased bone formation indices. Osteoblasts do not express Atp6v0d2 and its deletion did not affect osteoblast differentiation or expression of standard markers in vitro, indicating that the increase in formation is not due to intrinsic effects [83]. Specific investigation of OsteoMac contribution to osteopetrosis models is needed to clearly delineate all the cellular participants in ‘osteoblast coupling’. Conclusions and future directions Bone lining tissues are integral to the processes of bone modelling and remodelling, however neither the composition of cells within this

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tissue nor the phenotype of the cell generically referred to as the bone lining cell is well established. To further our understanding of bone biology, knowledge of the cellular constituents of osteal tissue is essential. Our data indicate that OsteoMacs are a significant population within the osteal microenvironment confirming that cells other than osteoblasts populate the bone surface. Macrophages are sentinel cells within tissues that coordinate development and repair through the expression and/or secretion of a wide range of regulatory molecules. Given the known trophic ability of macrophages and the anatomical location of OsteoMacs, we propose that these cells provide a new twist on the concept of osteoclast-to-osteoblast coupling, in that they, like their myeloid cousins the osteoclast, can facilitate osteoblast function and subsequently participate in the coordination of bone dynamics. References [1] Goldring SR. 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