Inducible transgenes under the control of the hCD68 promoter identifies mouse macrophages with a distribution that differs from the F4/80 - and CSF-1R-expressing populations

Abstract

Objective: Macrophages are critical components of diverse microenvironments (ME) in adulthood, as well as during embryogenesis. Their role in development precludes the use of gene-targeting and knockout approaches for studying their function. Hence, we proposed to create a macrophage-specific inducible transgenic mouse where genes can be turned on or off at will.

Materials and methods: A transgenic mouse in which the reverse tetracycline activator (rtTA-M2) is expressed under the hCD68 promoter for macrophage-specific gene induction was developed and crossed with a second transgenic reporter mouse strain in which the gene for green fluorescent protein (GFP) is under the control of tetracycline responsive element promoter. After doxycycline induction of the double transgenic animals (designated CD68-rtTA-tet-GFP), inducible expression of GFP was characterized by multicolor flow cytometric analysis of blood, marrow, and spleen cells and by demonstration of GFP expression in fresh-frozen sections in diverse tissues.

Results: In bone marrow, inducible GFP expression was not confined to, or inclusive of, all cells expressing the classical macrophage markers, such as F4/80. However, GFP-expressing cells in thioglycollate-elicited peritoneal macrophages were also positive for F4/80 and monocyte-macrophage-specific 2 antigen. Interestingly, flow analysis also indicated little overlap between the F4/80 and CSF-1R-positive populations. Fresh-frozen samples of tissues known to contain macrophages revealed GFP-expressing cells with variable morphologies.

Conclusion: Our results show that the hCD68 promoter directs gene expression in a macrophage population distinct from that defined by classical monocyte-macrophage markers or promoters. Whether this population is functionally distinct remains to be established.

Figure 1

A. The hCD68-rtTA-M2 construct. The 4.3 kbp construct includes the 2.9 Kbp genomic DNA immediately upstream of the start site, followed by the 83 bp fist intron (IVIS), the rtTA-M2, and the bovine growth hormone poly A tail. B. Demonstration of GFP in hematopoietic tissue of double transgenic animals induced with doxycycline (induced) or without doxycycline (control). BM – Bone Marrow, PB-Peripheral Blood, SPLN- Spleen. About 10% of all bone marrow cells were positive for GFP, while only 0.2 % of cells were positive in peripheral blood and 1% in spleen. Negligible numbers of cells were GFP-positive in the control animals. When GFP-positive cells were superimposed (green) onto FSC-SSC contour plots, they distributed to a pattern expected of monocytes-macrophages.

Figure 2. Identifying the lineage of the GFP-expressing cells in bone marrow by flow-cytometry

Gr-1 and Cd11b are markers of the myeloid lineage; the GFP-expressing cells were mostly contained in the Gr-1 and Cd11b fraction of cells. While most of the GFP-expressing cells also expressed F4/80, about 25% of GFP-expressing cells were F4/80-negative, suggesting that some mature macrophages are negative for F4/80 in hematopoietic tissue. The GFP-expressing cells were also analyzed for markers of other lineages including Thy1.2 (T-lymphocyte), B220 (B-lymphocyte) and Ter-119 (erythrocyte).

Figure 3

Panels A to C: Demonstration of GFP-expressing putative macrophages in thioglycollate elicited macrophages. These cells also express MOMA-2 (Panel B) as well as F4/80 (Panel C). Brief fixation (5 minutes) followed by permeabilization was performed prior to staining with MOMA-2. The fixation and permeabilization likely depleted the peritoneal cells of the neutrophils, and hence the proportion of GFP-expressing cells in Figures 3B (which were fixed and permeabilized for MOMA-2 staining) is higher than those shown in Figures 3A and 3C which are unfixed. Panels D to F: Demonstration of GFP-expressing putative macrophages in freshly frozen sections. Panels D and E: Thymus sections at 10X and 60X magnification, respectively. Thymic macrophages have intricate processes surrounding other macrophages. F and G: Kidney sections at 2X and 60X magnifications. Most of the GFP-expressing cells were found in the renal cortex and seen intercalated between renal tubules. Panel H: F4/80 and CSF-1R expression in murine bone marrow mononuclear cells (BMMNC). Wild-type C57/Bl6 mouse were analyzed for expression of F4/80 antigen and CSF-1R by multi-color flow cytometry results indicate that only a small proportion of BMMNC (1.5%) are CSF-1R-positive, while about 25% of cells are positive for F4/80. Amongst the CSF-1R-positive cells, 25% are negative for F4/80 antigen.