https://www.pure.ed.ac.uk/ws/files/14425897/Fate_Mapping_Reveals_Origins_and_Dynamics_of_Monocytes_and_Tissue_Macrophages_under_Homeostasis.pdf

Fate Mapping Reveals Origins and Dynamics of Monocytes and Tissue Macrophages under Homeostasis

Conditional mutagenesis in mice has recently been made possible through the combination of gene targeting techniques and site-directed mutagenesis, using the bacteriophage P1-derived Cre/loxP recombination system. The versatility of this approach depends on the availability of mouse mutants in which the recombinase Cre is expressed in the appropriate cell lineages or tissues. Here we report the generation of mice that express Cre in myeloid cells due to targeted insertion of the cre cDNA into their endogenous M lysozyme locus. In double mutant mice harboring both the LysMcre allele and one of two different loxP-flanked target genes tested, a deletion efficiency of 83-98% was determined in mature macrophages and near 100% in granulocytes. Partial deletion (16%) could be detected in CD11c+ splenic dendritic cells which are closely related to the monocyte/macrophage lineage. In contrast, no significant deletion was observed in tail DNA or purified T and B cells. Taken together, LysMcre mice allow for both specific and highly efficient Cre-mediated deletion of loxP-flanked target genes in myeloid cells.

Conditional gene targeting in macrophages and granulocytes using LysMcre mice.

Mice homozygous for the recessive mutation osteopetrosis (op) on chromosome 3 have a restricted capacity for bone remodelling, and are severely deficient in mature macrophages and osteoclasts. Both cell populations originate from a common haemopoietic progenitor. As op/op mice are not cured by transplants of normal bone marrow cells, the defects in op/op mice may be associated with an abnormal haematopoietic microenvironment rather than with an intrinsic defect in haematopoietic progenitors. To investigate the molecular and biochemical basis of the defects caused by the op mutation, we established primary fibroblast cell lines from op/op mice and tested the ability of these cell lines to support the proliferation of macrophage progenitors. We show that op/op fibroblasts are defective in production of functional macrophage colony-stimulating factor (M-CSF), although its messenger RNA (Csfm mRNA) is present at normal levels. This defect in M-CSF production and the recent mapping of the Csfm structural gene near op on chromosome 3 suggest that op is a mutation within the Csfm gene itself. We have sequenced Csfm complementary DNA prepared from op/op fibroblasts and found a single base pair insertion in the coding region of the Csfm gene that generates a stop codon 21 base pairs downstream. Thus, the op mutation is within the Csfm coding region and we conclude that the pathological changes in this mutant result from the absence of M-CSF.

The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene

Mononuclear phagocytes, including monocytes, macrophages, and dendritic cells, contribute to tissue integrity as well as to innate and adaptive immune defense. Emerging evidence for labor division indicates that manipulation of these cells could bear therapeutic potential. However, specific ontogenies of individual populations and the overall functional organization of this cellular network are not well defined. Here we report a fate-mapping study of the murine monocyte and macrophage compartment taking advantage of constitutive and conditional CX(3)CR1 promoter-driven Cre recombinase expression. We have demonstrated that major tissue-resident macrophage populations, including liver Kupffer cells and lung alveolar, splenic, and peritoneal macrophages, are established prior to birth and maintain themselves subsequently during adulthood independent of replenishment by blood monocytes. Furthermore, we have established that short-lived Ly6C(+) monocytes constitute obligatory steady-state precursors of blood-resident Ly6C(-) cells and that the abundance of Ly6C(+) blood monocytes dynamically controls the circulation lifespan of their progeny.

Fate Mapping Reveals Origins and Dynamics of Monocytes and Tissue Macrophages under Homeostasis (vol 38, pg 79, 2013)

The complementary DNAs and genes encoding the four major human myeloid growth factors--granulocyte colony-stimulating factor, macrophage colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and interleukin-3--have all been molecularly cloned. These DNA clones have proved valuable for studying the molecular biology of these important regulatory molecules as well as for the large-scale production of the recombinant growth factor proteins. These advances have led to a much better understanding of the role of the myeloid growth factors in regulating hematopoiesis in vivo that should soon find practical application in clinical medicine.

https://science.sciencemag.org/content/sci/236/4806/1229.full.pdf

The human hematopoietic colony-stimulating factors

https://www.cell.com/cell/pdf/0092-8674(82)90376-2.pdf

Survival of mononuclear phagocytes depends on a lineage-specific growth factor that the differentiated cells selectively destroy

CSF‐1—A mononuclear phagocyte lineage‐specific hemopoietic growth factor

The binding and uptake of the colony-stimulating factor CSF-1 by peritoneal exudate macrophages (PEM) from lipopolysaccharide insensitive C3H/HeJ mice was examined at 2 degrees C, and at 37 degrees C. At 2 degrees C, 125I-CSF-1 was bound irreversibly to the cell surface. At 37 degrees C, 90% of the cell surface associated 125I-CSF-1 was rapidly internalized and subsequently degraded and the remaining 10% dissociated as intact 125I-CSF-1. Thus classical equilibrium or steady state methods could not be used to quantitatively analyze ligand-cell interactions at either temperature, and alternative approaches were developed. At 2 degrees C, the equilibrium constant (Kd less than or equal to 10(-13) M) was derived from estimates of the rate constants for the binding (kon congruent to 8 x 10(5) M-1 s-1) and dissociation (koff less than or equal to 2 x 10(-7) s-1) reactions. At 37 degrees C, the processes of dissociation and internalization of bound ligand were kinetically competitive, and the data was formally treated as a system of competing first order reactions, yielding first order rate constants for dissociation, koff = 0.7 min-1 (t1/2 = 10 min) and internalization, kin = 0.07 min-1 (t 1/2 = 1 min). Approximately 15 min after internalization, low-molecular weight 125I-labeled degradation products began to appear in the medium. Release of this degraded 125I-CSF-1 was kinetically first order over three half-lives (Kd = 4.3 x 10(-2) min-1, t1/2 = 16 min). Thus CSF-1 binds to a single class of receptors on PEM, is internalized with a single rate limiting step, and is rapidly destroyed without segregation into more slowly degrading intracellular compartments.

Larry J. Guilbert

Papers

Survival of mononuclear phagocytes depends on a lineage-specific growth factor that the differentiated cells selectively destroy

CSF‐1—A mononuclear phagocyte lineage‐specific hemopoietic growth factor

Uptake and destruction of 125I-CSF-1 by peritoneal exudate macrophages.