Macrophages: Their Emerging Roles in Bone

Abstract

Macrophages are present in nearly all tissues and are critical for development, homeostasis, and regeneration. Resident tissue macrophages of bone, termed osteal macrophages, are recently classified myeloid cells that are distinct from osteoclasts. Osteal macrophages are located immediately adjacent to osteoblasts, regulate bone formation, and play diverse roles in skeletal homeostasis. Genetic or pharmacological modulation of macrophages in vivo results in significant bone phenotypes, and these phenotypes depend on which macrophage subsets are altered. Macrophages are also key mediators of osseous wound healing and fracture repair, with distinct roles at various stages of the repair process. A central function of macrophages is their phagocytic ability. Each day, billions of cells die in the body and efferocytosis (phagocytosis of apoptotic cells) is a critical process in both clearing dead cells and recruitment of replacement progenitor cells to maintain homeostasis. Recent data suggest a role for efferocytosis in bone biology and these new mechanisms are outlined. Finally, although macrophages have an established role in primary tumors, emerging evidence suggests that macrophages in bone support cancers which preferentially metastasize to the skeleton. Collectively, this developing area of osteoimmunology raises new questions and promises to provide novel insights into pathophysiologic conditions as well as therapeutic and regenerative approaches vital for skeletal health.

Keywords: BONE FORMATION; EFFEROCYTOSIS; FRACTURE REPAIR; MACROPHAGE; SKELETAL METASTASIS.

Fig. 1

(A–C) Immunohistochemical staining (brown) of macrophages (F4/80⁺) in 4-week-old C57/B6 mice highlight their location in cortical and trabecular bone, and location immediately next to osteoblasts. (A) Macrophages (F4/80⁺) are located on the periosteal and endosteal surfaces of cortical bone, as well as in the marrow space. (B) Macrophages are located immediately adjacent to osteoblasts and line the bone formation surface, as shown here on the endosteal surface. This image was co-stained with TRAP (red stain) to mark osteoclasts and shows that despite sharing same lineage, TRAP+ cells (red arrows) are not F4/80⁺, and vice versa. (C) Similar to cortical bone, macrophages are also present throughout the trabecular secondary spongiosa (black arrows highlight examples of several positive cells). The distal growth plate is located immediately above the pictured field of view. (D–F) The goal of these images are to highlight the efferocytosis process of an apoptotic osteoblastic cell from initial apoptotic cell recognition by the macrophage to total cell engulfment. Primary murine macrophages were cultured for 7 days with M-CSF and stained green with CFSE. Osteoblastic (MC4) cells were stained deep red, and subsequently induced to undergo apoptosis with UV light (30 min). Deep red–stained apoptotic osteoblast-like cells were then cocultured with green macrophages, which underwent efferocytosis of the apoptotic osteoblastic cells over 5 to 10 hours. The efferocytosis process is highlighted, showing initial macrophage recognition of an apoptotic osteoblastic cell (D), engulfment (E), and finally an apoptotic osteoblastic cell totally engulfed by a macrophage (F). CFSE = carboxyfluorescein succinimidyl ester.

Fig. 2

Osteal macrophages are located on the bone surface and found immediately adjacent to osteoblasts and support bone formation. Intermittent PTH (iPTH) treatment has been shown to induce osteoblastic expression of factors such as IL-6 and sIL-6r, which support expansion of the myeloid cell population (osteal macrophage and osteoclast precursors). The question mark indicates the relatively unknown source of osteal macrophages on the bone surface. At the end of an osteoblast life cycle it has three fates: (1) ~15% become embedded in the bone matrix as osteocytes; (2) ~30% become quiescent bone lining cells; and (3) the remaining ~40% to 70% likely die by apoptosis. Apoptotic osteoblasts are efficiently cleared by macrophages in a process called efferocytosis. The engulfment process of efferocytosis facilitated by expression of “eat-me” signals including PS on apoptotic cells, which are attached to macrophage proteins such as α_vβ₃ or Mer by linking proteins MFG-E8 or Gas6. Other efferocytosis signals have been identified but are not depicted here. Moreover, the efferocytosis process is generally associated with macrophage production of specific proteins such as TGF-β. These factors may facilitate continued bone modeling by replenishing the osteoblast population from progenitor cells. OB = osteoblast; OC = osteoclast; IL-6 = interleukin-6; sIL-6R = soluble interleukin-6 receptor; M-CSF = macrophage colony stimulating factor, RANK(L) = receptor activator of nuclear factor κB (ligand); TGF-β = transforming growth factor beta; PS = phosphatidylserine; MFG-8 = milk fat globule-EGF factor 8; α_vβ₃= alpha-V beta-3 integrin; Gas6 = growth arrest-specific 6; MER(tk) = receptor tyrosine kinase MerTK; MSCs = mesenchymal stromal cells.