Unique Gene Expression Profiles During Osteal Macrophage Efferocytosis

Objectives: Apoptotic cell clearance by macrophages, termed efferocytosis, is vital for development, homeostasis, and repair of many tissues, however the role of this process in bone is incompletely understood. Apoptosis is a frequent and natural fate of bone cells during homeostasis, and causes a phagocytic response in macrophages. This study aimed to identify gene expression unique to osteal macrophages when undergoing efferocytosis of apoptotic bone cells versus non-bone cells.
Methods: Primary bone marrow-cells derived from 6-8 week old C57BL/6 mice were cultured 1wk with M-CSF to derive macrophages. Murine primary bone marrow stromal cells (BMSCs) and thymocytes (thym) were isolated and UV irradiated to induce apoptosis. Apoptotic BMSCs or thym cells were then added to macrophages in co-culture (macs+apBMSCs or macs+apThym), and after 6hrs of allowing macrophages to efferocytose either cell type, RNA was isolated. An Affymetrix Mouse Gene 2.1 ST Array was analyzed for differences in gene expression of efferocytic versus control macrophages.
Results: An initial screen for significant gene changes revealed 1187 genes uniquely regulated in macs+apBMSCs. Far fewer genes (101) were unique to macs+apThym co-cultures, and only 32 genes were commonly regulated in both cell types. For example, the anabolic factor fibroblast growth factor2 (Fgf2), was 3.76 fold higher in macs+apBMSCs vs. macrophages alone, but not increased when macrophages efferocytosed thymocytes, which was verified by polymerase chain reaction. These data suggest the response was specific to the cell type introduced. Furthermore, receptor activator of nuclear factor kappa-β ligand (Rankl) was 5.69-fold up-regulated in efferocytotic macrophages with BMSCs and not thymocytes.
Conclusions: Given the large difference in the genes expressed by macrophages when exposed to apoptotic BMSCs versus thymocytes, data suggest that bone marrow macrophages respond uniquely to different apoptotic cells in the bone marrow. Taken together, up-regulation of Fgf2 and Rankl by macrophages phagocytosing apoptotic BMSCs suggests that efferocytosis signals bone turnover. The specific response of macrophages to apoptotic BMSCs supports a critical role for efferocytosis in bone homeostasis.