Lindsey Moffat

We constructed a series of recombinant genomes which directed expression of the enzyme chloramphenicol acetyltransferase (CAT) in mammalian cells. The prototype recombinant in this series, pSV2-cat, consisted of the beta-lactamase gene and origin of replication from pBR322 coupled to a simian virus 40 (SV40) early transcription region into which CAT coding sequences were inserted. Readily measured levels of CAT accumulated within 48 h after the introduction of pSV2-cat DNA into African green monkey kidney CV-1 cells. Because endogenous CAT activity is not present in CV-1 or other mammalian cells, and because rapid, sensitive assays for CAT activity are available, these recombinants provided a uniquely convenient system for monitoring the expression of foreign DNAs in tissue culture cells. To demonstrate the usefulness of this system, we constructed derivatives of pSV2-cat from which part or all of the SV40 promoter region was removed. Deletion of one copy of the 72-base-pair repeat sequence in the SV40 promoter caused no significant decrease in CAT synthesis in monkey kidney CV-1 cells; however, an additional deletion of 50 base pairs from the second copy of the repeats reduced CAT synthesis to 11% of its level in the wild type. We also constructed a recombinant, pSV0-cat, in which the entire SV40 promoter region was removed and a unique HindIII site was substituted for the insertion of other promoter sequences.

We have produced an Fc conjugate of colony-stimulating factor (CSF) 1 with an improved circulating half-life. CSF1-Fc retained its macrophage growth-promoting activity, and did not induce proinflammatory cytokines in vitro. Treatment with CSF1-Fc did not produce adverse effects in mice or pigs. The impact of CSF1-Fc was examined using the Csf1r-enhanced green fluorescent protein (EGFP) reporter gene in MacGreen mice. Administration of CSF1-Fc to mice drove extensive infiltration of all tissues by Csf1r-EGFP positive macrophages. The main consequence was hepatosplenomegaly, associated with proliferation of hepatocytes. Expression profiles of the liver indicated that infiltrating macrophages produced candidate mediators of hepatocyte proliferation including urokinase, tumor necrosis factor, and interleukin 6. CSF1-Fc also promoted osteoclastogenesis and produced pleiotropic effects on other organ systems, notably the testis, where CSF1-dependent macrophages have been implicated in homeostasis. However, it did not affect other putative CSF1 targets, notably intestine, where Paneth cell numbers and villus architecture were unchanged. CSF1 has therapeutic potential in regenerative medicine in multiple organs. We suggest that the CSF1-Fc conjugate retains this potential, and may permit daily delivery by injection rather than continuous infusion required for the core molecule.

We investigated the role of CSF1R signaling in adult mice using prolonged treatment with anti-CSF1R antibody. Mutation of the CSF1 gene in the op/op mouse produces numerous developmental abnormalities. Mutation of the CSF1R has an even more penetrant phenotype, including perinatal lethality, because of the existence of a second ligand, IL-34. These effects on development provide limited insight into functions of CSF1R signaling in adult homeostasis. The carcass weight and weight of several organs (spleen, kidney, and liver) were reduced in the treated mice, but overall body weight gain was increased. Despite the complete loss of Kupffer cells, there was no effect on liver gene expression. The treatment ablated OCL, increased bone density and trabecular volume, and prevented the decline in bone mass seen in female mice with age. The op/op mouse has a deficiency in pancreatic β cells and in Paneth cells in the gut wall. Only the latter was reproduced by the antibody treatment and was associated with increased goblet cell number but no change in villus architecture. Male op/op mice are infertile as a result of testosterone insufficiency. Anti-CSF1R treatment ablated interstitial macrophages in the testis, but there was no sustained effect on testosterone or LH. The results indicate an ongoing requirement for CSF1R signaling in macrophage and OCL homeostasis but indicate that most effects of CSF1 and CSF1R mutations are due to effects on development.

Testosterone synthesis depends on normal Leydig cell (LC) development, but the mechanisms controlling this development remain unclear. We recently demonstrated that androgen receptor (AR) ablation from a proportion of testicular peritubular myoid cells (PTM-ARKO) did not affect LC number, but resulted in compensated LC failure. The current study extends these investigations, demonstrating that PTM AR signalling is important for normal development, ultrastructure and function of adult LCs. Notably, mRNAs for LC markers [e.g. steroidogenic factor 1 (Nr5a1), insulin-like growth factor (Igf-1) and insulin-like factor 3 (Insl3)] were significantly reduced in adult PTM-ARKOs, but not all LCs were similarly affected. Two LC sub-populations were identified, one apparently 'normal' sub-population that expressed adult LC markers and steroidogenic enzymes as in controls, and another 'abnormal' sub-population that had arrested development and only weakly expressed INSL3, luteinizing hormone receptor, and several steroidogenic enzymes. Furthermore, unlike 'normal' LCs in PTM-ARKOs, the 'abnormal' LCs did not involute as expected in response to exogenous testosterone. Differential function of these LC sub-populations is likely to mean that the 'normal' LCs work harder to compensate for the 'abnormal' LCs to maintain normal serum testosterone. These findings reveal new paracrine mechanisms underlying adult LC development, which can be further investigated using PTM-ARKOs.

The MacBlue transgenic mouse uses the Csf1r promoter and first intron to drive expression of gal4-VP16, which in turn drives a cointegrated gal4-responsive UAS-ECFP cassette. The Csf1r promoter region used contains a deletion of a 150 bp conserved region covering trophoblast and osteoclast-specific transcription start sites. In this study, we examined expression of the transgene in embryos and adult mice. In embryos, ECFP was expressed in the large majority of macrophages derived from the yolk sac, and as the liver became a major site of monocytopoiesis. In adults, ECFP was detected at high levels in both Ly6C+ and Ly6C- monocytes and distinguished them from Ly6C+, F4/80+, CSF1R+ immature myeloid cells in peripheral blood. ECFP was also detected in the large majority of microglia and Langerhans cells. However, expression was lost from the majority of tissue macrophages, including Kupffer cells in the liver and F4/80+ macrophages of the lung, kidney, spleen and intestine. The small numbers of positive cells isolated from the liver resembled blood monocytes. In the gut, ECFP+ cells were identified primarily as classical dendritic cells or blood monocytes in disaggregated cell preparations. Immunohistochemistry showed large numbers of ECFP+ cells in the Peyer's patch and isolated lymphoid follicles. The MacBlue transgene was used to investigate the effect of treatment with CSF1-Fc, a form of the growth factor with longer half-life and efficacy. CSF1-Fc massively expanded both the immature myeloid cell (ECFP-) and Ly6C+ monocyte populations, but had a smaller effect on Ly6C- monocytes. There were proportional increases in ECFP+ cells detected in lung and liver, consistent with monocyte infiltration, but no generation of ECFP+ Kupffer cells. In the gut, there was selective infiltration of large numbers of cells into the lamina propria and Peyer's patches. We discuss the use of the MacBlue transgene as a marker of monocyte/macrophage/dendritic cell differentiation.