Mature monocytic cells enter tissues and engraft

Abstract

The goal of this study was to identify the circulating cell that is the immediate precursor of tissue macrophages. ROSA 26 marrow mononuclear cells (containing the beta-geo transgene that encodes beta-galactosidase and neomycin resistance activities) were cultured in the presence of macrophage colony-stimulating factor and flt3 Ligand for 6 days to generate monocytic cells at all stages of maturation. Expanded monocyte cells (EMC), the immature (ER-MP12(+)) and more mature (ER-MP20(+)) subpopulations, were transplanted into irradiated B6/129 F2 mice. beta-gal staining of tissue sections from animals 15 min after transplantation demonstrated that the donor cells landed randomly. By 3 h, donor cells in lung and liver were more frequent in animals transplanted with ER-MP20(+) (more mature) EMC than in animals transplanted with unseparated EMC or fresh marrow mononuclear cells, a pattern that persisted at 3 and 7 days. At 3 days, donor cells were found in spleen, liver, lung, and brain (rarely) as clusters as well as individual cells. By 7 and 14 days, the clusters had increased in size, and the cells expressed the macrophage antigen F4/80, suggesting that further replication and differentiation had occurred. PCR for the neogene was used to quantitate the amount of donor DNA in tissues from transplanted animals and confirmed that ER-MP20(+) EMC preferentially engrafted. These data demonstrate that a mature monocytic cell gives rise to tissue macrophages. Because these cells can be expanded and manipulated in vitro, they may be a suitable target population for gene therapy of lysosomal storage diseases.

Figure 1

Diagram of antigen expression during monocyte and macrophage differentiation. Adapted in part from ref. .

Figure 2

β-gal⁺ donor cell landing in brain (A), lung (B), liver (C), and spleen (D) after transplantation. Fresh marrow mononuclear cells, EMC, or EMC fractionated into ER-MP20⁺, ER-MP12⁺, and ER-MP20⁻12⁺ subpopulations were transplanted into B6/129F2 mice. Organs were removed at various time points and frozen tissue sections were prepared and studied for the presence of β-gal⁺ cells. Data are presented as the average number of β-gal⁺ cells per tissue section (y axis).

Figure 3

Engraftment of expanded monocytic cells in lung and brain after transplantation. β-Gal staining of cryostat sections of tissues from B6/129F2 animals transplanted with M-CSF- and FL-expanded ROSA 26 macrophage precursors are shown. A and B are sections from lung demonstrating a doublet of β-gal⁺ (blue) cells 3 days after transplantation (A) and a larger cluster of β-gal⁺ cells at 7 days (B). Rare groups of β-gal⁺ cells were seen in the brain of an animal at 14 days (C, arrows).

Figure 4

neo⁺ donor cell landing in various brain (A), lung (B), liver (C), and spleen (D) after transplantation. Fresh marrow mononuclear cells, EMC, or EMC fractionated into ER-MP20⁺, ER-MP12⁺, and ER-MP20⁻12⁺ subpopulations were transplanted into B6/129F2 mice. Organs were removed at various time points, and genomic DNA was extracted and studied for the presence of neo⁺ cells by using a semiquantitative PCR. Data are presented as the average percentage of neo⁺ cells per tissue sample (y axis).