Developmental and functional significance of the CSF-1 proteoglycan chondroitin sulfate chain

The primary macrophage growth factor, colony-stimulating factor-1 (CSF-1), is homodimeric and exists in 3 biologically active isoforms: a membrane-spanning, cell-surface glycoprotein (csCSF-1) and secreted glycoprotein (sgCSF-1) and proteoglycan (spCSF-1) isoforms. To investigate the in vivo role of the chondroitin sulfate glycosaminoglycan (GAG) chain of spCSF-1, we created mice that exclusively express, in a normal tissue-specific and developmental manner, either the secreted precursor of spCSF-1 or the corresponding precursor in which the GAG addition site was mutated. The reproductive, hematopoietic tooth eruption and tissue macrophage defects of CSF-1-deficient, osteopetrotic Csf1(op)/Csf1(op) mice were corrected by transgenic expression of the precursors of either sgCSF-1 or spCSF-1. Furthermore, in contrast to the transgene encoding csCSF-1, both failed to completely correct growth retardation, suggesting a role for csCSF-1 in the regulation of body weight. However, spCSF-1, in contrast to sgCSF-1, completely resolved the osteopetrotic phenotype. Furthermore, in transgenic lines expressing different concentrations of sgCSF-1 or spCSF-1, spCSF-1 more efficiently corrected Csf1(op)/Csf1(op) defects of tooth eruption, eyelid opening, macrophage morphology, and B-cell deficiency than sgCSF-1. These results indicate an important role of the CSF-1 chondroitin sulfate proteoglycan in in vivo signaling by secreted CSF-1.     

Rescue of the colony-stimulating factor 1 (CSF-1)-nullizygous mouse (Csf1(op)/Csf1(op)) phenotype with a CSF-1 transgene and identification of sites of local CSF-1 synthesis.

Colony-stimulating factor 1 (CSF-1) regulates the survival, proliferation, and differentiation of mononuclear phagocytes. It is expressed as a secreted glycoprotein or proteoglycan found in the circulation or as a biologically active cell-surface glycoprotein. To investigate tissue CSF-1 regulation, CSF-1-null Csf1(op)/Csf1(op) mice expressing transgenes encoding the full-length membrane-spanning CSF-1 precursor driven by 3.13 kilobases of the mouse CSF-1 promoter and first intron were characterized. Transgene expression corrected the gross osteopetrotic, neurologic, weight, tooth, and reproductive defects of Csf1(op)/Csf1(op) mice. Detailed analysis of one transgenic line revealed that circulating CSF-1, tissue macrophage numbers, hematopoietic tissue cellularity, and hematopoietic parameters were normalized. Tissue CSF-1 levels were normal except for elevations in 4 secretory tissues. Skin fibroblasts from the transgenic mice secreted normal amounts of CSF-1 but also expressed some cell-surface CSF-1. Also, lacZ driven by the same promoter/first intron revealed beta-galactosidase expression in hematopoietic, reproductive, and other tissue locations proximal to CSF-1 cellular targets, consistent with local regulation by CSF-1 at these sites. These studies indicate that the 3.13-kilobase promoter/first intron confers essentially normal CSF-1 expression. They also pinpoint new cellular sites of CSF-1 expression, including ovarian granulosa cells, mammary ductal epithelium, testicular Leydig cells, serous acinar cells of salivary gland, Paneth cells of the small intestine, as well as local sites in several other tissues.     

Colony-stimulating factor 1 regulation of neuroendocrine pathways that control gonadal function in mice

Colony stimulating factor 1 (CSF-1) is the primary regulator of cells of the mononuclear phagocytic lineage. Consequently mice lacking CSF-1 (Csf1(op)/Csf1(op)) have depleted populations of macrophages in many tissues. In addition, both sexes have reduced fertility with females having extended estrus cycles and poor ovulation rates, whereas males have low circulating LH and T. In this study, we show that puberty was significantly delayed in Csf1(op)/Csf1(op) females compared with control littermates. Restoration of circulating CSF-1 over the first 2 wk of life accelerated puberty, and this treatment until puberty completely corrected the extended estrous cycles. In a standard LH surge induction protocol, Csf1(op)/Csf1(op) females showed diminutive negative and no positive feedback response to E2. These data, together with that from male Csf1(op)/Csf1(op) mice that showed normal release of LH with a GnRH agonist, indicate that the hypothalamus is the site of the primary defect causing fertility problems in CSF-1-deficient mice. In the hypothalamus, microglia are the only CSF-1 receptor-bearing cells, and the recruitment of a full complement these cells is slightly delayed in Csf1(op)/Csf1(op) mice. These data suggest a role for CSF-1 and its target cells, microglia, in establishing the feedback sensitivity to circulating steroid hormones in the hypothalamus of mice.     

Targeted disruption of the mouse colony-stimulating factor 1 receptor gene results in osteopetrosis, mononuclear phagocyte deficiency, increased primitive progenitor cell frequencies, and reproductive defects.

The effects of colony-stimulating factor 1 (CSF-1), the primary regulator of mononuclear phagocyte production, are thought to be mediated by the CSF-1 receptor (CSF-1R), encoded by the c-fms proto-oncogene. To investigate the in vivo specificity of CSF-1 for the CSF-1R, the mouse Csf1r gene was inactivated. The phenotype of Csf1(-)/Csf1r(-) mice closely resembled the phenotype of CSF-1-nullizygous (Csf1(op)/Csf1(op)) mice, including the osteopetrotic, hematopoietic, tissue macrophage, and reproductive phenotypes. Compared with their wild-type littermates, splenic erythroid burst-forming unit and high-proliferative potential colony-forming cell levels in both Csf1(op)/Csf1(op) and Csf1(-)/Csf1r(-) mice were significantly elevated, consistent with a negative regulatory role of CSF-1 in erythropoiesis and the maintenance of primitive hematopoietic progenitor cells. The circulating CSF-1 concentration in Csf1r(-)/Csf1r(-) mice was elevated 20-fold, in agreement with the previously reported clearance of circulating CSF-1 by CSF-1R-mediated endocytosis and intracellular destruction. Despite their overall similarity, several phenotypic characteristics of the Csf1r(-)/Csf1r(-) mice were more severe than those of the Csf1(op)/Csf1(op) mice. The results indicate that all of the effects of CSF-1 are mediated via the CSF-1R, but that subtle effects of the CSF-1R could result from its CSF-1-independent activation.     

CSF-1 deficiency in the op/op mouse has differential effects on macrophage populations and differentiation stages.

Osteopetrosis and the absence of colony-stimulating factor 1 (CSF-1) in op/op mice are associated with decreased cellularity of the bone marrow (to one tenth of the normal), a very significant reduction in the number of cells recovered from peritoneal, pleural, and alveolar lavages, moderate leukopenia, and a slight decrease in the number of cells per spleen and thymus. Furthermore, op/op mice possess deficiencies in the number of macrophages in various organs. These cells are apparently absent in the bone marrow, severely reduced (5%-15% of the normal number) in peritoneal and pleural cavities and in the lungs. In addition, a marked decrease in the frequency and total number of circulating monocytes is present (5% of the normal). The deficiency of macrophages is less severe in the liver, spleen, and thymus of op/op mice (approximately 30% of those seen in normal). There is a concomitant redistribution of macrophage progenitor cells (granulocyte-macrophage colony-forming units, CFU-GM) in op/op mice from the marrow to the spleen and liver, associated with an increased sensitivity to interleukin 3 (IL-3). Their total number is decreased at least threefold compared to control mice. Moreover, op/op mice have at least a fivefold reduction in the total number of day-11 spleen colony-forming units (CFU-S) associated with their redistribution to the spleen and liver. These data suggest that the macrophage system in op/op mice is reduced at all levels tested, that is, at the level of mature macrophages, the level of progenitors, and the level of stem cells, whereas the redistribution of progenitor and stem cells could be viewed as a secondary consequence of osteopetrosis. Furthermore, these data suggest that macrophage dependency in vivo on CSF-1 is limited and different in various organs. Particularly in the liver, spleen, and thymus, other growth factors may significantly compensate for CSF-1 deficiency. Based on the relative decrease in the number of CFU-GM in the op/op mice, it appears that the population size of these progenitors is less dependent on CSF-1 than the hematopoietic stem cell population size as evidenced by the day-11 CFU-S assay. The day-11 CFU-S population is severely reduced in op/op mice, suggesting a physiological involvement of CSF-1 in expanding its size. These data provide evidence that CSF-1, besides acting on the final and intermediate stages of macrophage maturation, may also play a role in early stages of hematopoiesis.     

Regulation of cell survival during B lymphopoiesis in mouse bone marrow: enhanced pre-B-cell apoptosis in CSF-1-deficient op/op mutant mice

OBJECTIVE: Osteopetrotic (op/op) mice are deficient in macrophages and osteoclasts due to a CSF-1 gene mutation. The aim of this study was to evaluate the effect of these deficiencies and of CSF-1-dependent mechanisms on B lymphopoiesis in bone marrow, with special reference to the apoptotic activity of precursor B cells. MATERIALS AND METHODS: B-cell development and apoptosis were examined in the bone marrow of op/op mice using immunofluorescence labeling and flow cytometry. Short-term cultures of bone marrow were used to evaluate the effect of recombinant CSF-1 on the rate of B-cell apoptosis. RESULTS: Bone marrow cellularity was greatly reduced in op/op mice compared with normal littermates. However, precursor B cells were disproportionately decreased, most markedly at the pre-B-cell stage. Precursor B cells, particularly pre-B cells, displayed elevated apoptotic incidences both ex vivo and in short-term culture. Addition of recombinant CSF-1 reduced the incidence of apoptosis among precursor B cells in short-term cultures of whole bone marrow suspensions from normal mice but not in cultures of sorted B220+ B-lineage cells. CONCLUSIONS: The finding of increased pre-B-cell apoptosis in op/op mice provides evidence that CSF-1-dependent mechanisms can strongly influence the survival of precursor B cells in mouse bone marrow, particularly at the pro-B/pre-B cell transition. It is proposed that the local or systemic levels of CSF-1 during ontogeny may thus play a role in regulating B-cell production within the bone marrow microenvironment.     

Immortalized fibroblastoid cells of osteopetrotic mutant mice (op/op) do not secrete CSF-1 and do not inhibit CSF-1 activity released by normal cells

Osteopetrotic mutant mice (op/op) are deficient in osteoclasts and macrophages. In this report, the establishment of clonal permanent fibroblastoid cell lines from spleens of op/+ and op/op mice is described and conditioned medium obtained from these cell lines examined for release of growth factor that stimulates the proliferation and differentiation of macrophage precursor cells. It is shown that conditioned medium of op/+ fibroblastoid cells contains CSF-1 activity, whereas conditioned medium of op/op fibroblastoid cells does not. Conditioned medium of op/op cell lines does not inhibit colony-stimulating factor 1 (CSF-1) activity in conditioned medium of op/+ cells. Hybrids between op/op and op/+ fibroblastoid cells release CSF-1 activity in amounts similar to op/+ cell lines alone. These results confirm that op/op mice do not produce any CSF-1 activity. The data further suggest that predicted truncated proteins expressed from the mutated CSF-1 gene in op/op mice are functionally not relevant and that there is no evidence for trans-acting factors in op/op that might inhibit CSF-1 expression.     

Spleen serves as a reservoir of osteoclast precursors through vitamin D-induced IL-34 expression in osteopetrotic op/op mice

Osteoclasts are generated from monocyte/macrophage-lineage precursors in response to colony-stimulating factor 1 (CSF-1) and receptor activator of nuclear factor-κB ligand (RANKL). CSF-1-mutated CSF-1(op/op) mice as well as RANKL(-/-) mice exhibit osteopetrosis (OP) caused by osteoclast deficiency. We previously identified RANKL receptor (RANK)/CSF-1 receptor (CSF-1R) double-positive cells as osteoclast precursors (OCPs), which existed in bone in RANKL(-/-) mice. Here we show that OCPs do not exist in bone but in spleen in CSF-1(op/op) mice, and spleen acts as their reservoir. IL-34, a newly discovered CSF-1R ligand, was highly expressed in vascular endothelial cells in spleen in CSF-1(op/op) mice. Vascular endothelial cells in bone also expressed IL-34, but its expression level was much lower than in spleen, suggesting a role of IL-34 in the splenic generation of OCPs. Splenectomy (SPX) blocked CSF-1-induced osteoclastogenesis in CSF-1(op/op) mice. Osteoclasts appeared in aged CSF-1(op/op) mice with up-regulation of IL-34 expression in spleen and bone. Splenectomy blocked the age-associated appearance of osteoclasts. The injection of 2-methylene-19-nor-(20S)-1α,25(OH)(2)D(3) (2MD), a potent analog of 1α,25-dihidroxyvitamin D(3), into CSF-1(op/op) mice induced both hypercalcemia and osteoclastogenesis. Administration of 2MD enhanced IL-34 expression not only in spleen but also in bone through a vitamin D receptor-mediated mechanism. Either splenectomy or siRNA-mediated knockdown of IL-34 suppressed 2MD-induced osteoclastogenesis. These results suggest that IL-34 plays a pivotal role in maintaining the splenic reservoir of OCPs, which are transferred to bone in response to diverse stimuli, in CSF-1(op/op) mice. The present study also suggests that the IL-34 gene in vascular endothelial cells is a unique target of vitamin D.     

Total absence of colony-stimulating factor 1 in the macrophage-deficient osteopetrotic (op/op) mouse.

Osteopetrotic (op/op) mutant mice suffer from congenital osteopetrosis due to a severe deficiency of osteoclasts. Furthermore, the total number of mononuclear phagocytes is extremely low in affected mice. Serum, 11 tissues, and different cell and organ conditioned media from op/op mice were shown to be devoid of biologically active colony-stimulating factor 1 (CSF-1), whereas all of these preparations from littermate control +/+ and +/op mice contained the growth factor. The deficiency was specific for CSF-1 in that serum or conditioned media from op/op mice possessed elevated levels of at least three other macrophage growth factors. Partial correction of the op/op defect was observed following intraperitoneal implantation of diffusion chambers containing L929 cells, which in culture produce CSF-1 as their sole macrophage growth factor. No rearrangement of the CSF-1 gene in op/op mice was detected by Southern analysis. However, in contrast to control lung fibroblasts, which contained 4.6- and 2.3-kilobase CSF-1 mRNAs, only the 4.6-kilobase species was detected in op/op cells. An alteration in the CSF-1 gene is strongly implicated as the primary defect in op/op mice because they do not contain detectable CSF-1, their defect is correctable by administration of CSF-1, the op locus and the CSF-1 gene map within the same region of mouse chromosome 3, their CSF-1 mRNA biosynthesis is altered, and the op/op phenotype is consistent with the phenotype expected in a CSF-1 deficient mouse.     

Correction by CSF-1 of defects in the osteopetrotic op/op mouse suggests local, developmental, and humoral requirements for this growth factor

Mice that are mutant at the op locus have a severe deficiency of mononuclear phagocytes due to an inactivating mutation in the CSF-1 (macrophage colony-stimulating factor, M-CSF) gene. op/op mice are toothless, possessing skeletal abnormalities, a low body weight, and compromised fertility; they are osteopetrotic due to a deficiency of osteoclasts. The congenital osteopetrosis, toothless phenotype, osteoclast deficit, and the defects in splenic and femoral macrophages were corrected by routes of administration of human recombinant CSF-1 that maintained normal circulating CSF-1 concentrations. Early restoration of circulating CSF-1 was required for rescue of the toothless phenotype, but only partially restored body weight. In contrast, the deficiencies of pleural and peritoneal cavity macrophages and the reduced female fertility were not corrected by restoration of circulating CSF-1. These results suggest that although circulating CSF-1 is required for osteoclast and macrophage production, local synthesis and action of the growth factor are important for certain target cell populations.     

VEGF receptor 1 signaling is essential for osteoclast development and bone marrow formation in colony-stimulating factor 1-deficient mice

VEGF receptor 1 (VEGFR-1/Flt-1) is a high-affinity tyrosine kinase (TK) receptor for VEGF and regulates angiogenesis as well as monocyte/macrophage functions. We previously showed that the osteoclast deficiency in osteopetrotic Csf1op/Csf1op (op/op) mice is gradually restored in an endogenous, VEGF-dependent manner. However, the molecular basis of the recovery is still not clear. To examine which VEGFR is important and to clarify how colony-stimulating factor 1 (CSF-1) and VEGF signals interact in osteoclastogenesis, we introduced a VEGFR-1 signaling deficiency (Flt1(TK)-/-) into op/op mice. The original Flt1(TK)-/- mice showed mild osteoclast reduction without bone marrow suppression. The double mutant (op/opFlt1(TK)-/-) mice, however, exhibited very severe osteoclast deficiency and did not have numbers of osteoclasts sufficient to form the bone marrow cavity. The narrow bone marrow cavity in the op/opFlt1(TK)-/- mice was gradually replaced with fibrous tissue, resulting in severe marrow hypoplasia and extramedullary hematopoiesis. In addition to osteoclasts, osteoblasts also decreased in number in the op/opFlt1(TK)-/- mice. These results strongly suggest that the interaction of signals by means of VEGFR-1 and the CSF-1 receptor plays a predominant role not only in osteoclastogenesis but also in the maintenance of bone marrow functions.     

Rescue of the osteopetrotic defect in op/op mice by osteoblast-specific targeting of soluble colony-stimulating factor-1

Soluble colony-stimulating factor-1 (sCSF-1) and membrane bound CSF-1 are synthesized by osteoblasts and stromal cells. However, the precise role of each form in osteoclastogenesis is unclear. In the op/op mouse, absence of osteoblast-derived CSF-1 leads to decreased osteoclasts and osteopetrosis. To determine whether sCSF-1 gene replacement can cure the osteopetrotic defect, we took advantage of the osteoblast specificity of the osteocalcin promoter to selectively express sCSF-1 in the bone of op/op mice. Transgenic mice harboring the human sCSF-1 cDNA under the control of the osteocalcin promoter were generated and cross-bred with heterozygous op/wt mice to establish op/op mutants expressing the transgene (op/opT). The op/op genotype and transgene expression were confirmed by PCR and Southern blot analysis, respectively. High levels of human sCSF-1 protein were selectively expressed in bone. At 2(1/2) wk, op/opT mice showed normal growth and tooth eruption. Femurs removed at 5 and 14 wk were analyzed by peripheral quantitative computed tomography and histomorphometry. The abnormal bone mineral density, cancellous bone volume, and growth plate width observed in op/op mice was completely reversed in op/opT mice by 5 wk, and this effect persisted at 14 wk, with measurements comparable with wt/wt mice at each time point. Correction of the skeletal abnormalities in the 5-wk-old op/opT mice correlated with a marked increase in the total osteoclast number, and their number per millimeter of bone surface compared with that of op/op mutants. Osteoclast number was maintained at 14 wk in op/opT mice and morphologically resembled wt/wt osteoclasts. These results indicate that sCSF-1 is sufficient to drive normal osteoclast development and that the osteocalcin promoter provides an efficient tool for delivery of exogenous genes to the bone. Moreover, targeting sCSF-1 to osteoblasts in the bone microenvironment may be a potentially useful therapeutic modality for treating bone disorders.     

The cell surface form of colony-stimulating factor-1 is biologically active in bone in vivo

The specific biological function of the cell surface or membrane-bound isoform of colony-stimulating factor-1 (mCSF-1) is not well understood. To help define the role of this isoform in bone, we developed a transgenic mouse in which targeted expression of human mCSF-1 in osteoblasts was achieved under the control of the 2.4-kb rat collagen type I alpha promoter. Bone density, determined by peripheral quantitative computed tomography, was reduced 7% in mCSF-1 transgenic compared with that in wild-type mice. Histomorphometric analyses indicated that the number of osteoclasts in bone (NOc/BPm, NOc/TAR, OcS/BS) was significantly increased in transgenic mice (1.7- to 1.8-fold; P < 0.05 to P < 0.01) compared with that in wild-type animals. Interestingly, the osteoblast-restricted isoform transgene corrected the osteopetrosis seen in CSF-1-deficient op/op mice. Skeletal growth and bone density in op/op mice expressing mCSF-1 in osteoblasts were similar to those in wild-type mice and were dramatically different from those in the unmanipulated op/op animals. The op/op mice expressing mCSF-1 in bone had normal incisor and molar tooth eruption, whereas the op/op mice evidenced the expected failure of tooth eruption. These findings directly support the conclusion that mCSF-1 is functionally active in bone in vivo and is probably an important local source of CSF-1.     

Lipopolysaccharide-induced cytokine and receptor expression and neutrophil infiltration in the liver of osteopetrosis (op/op) mutant mice

BACKGROUND/AIMS: Mice homozygous for the osteopetrosis (op) mutation are genetically deficient in macrophage colony-stimulating factor (M-CSF/CSF-1) and are characterized by defective differentiation and function of macrophages. The aim of this study is to assess the contribution of M-CSF to lipopolysaccharide (LPS)-induced cytokine expression and neutrophil infiltration in the liver. Methods: We investigated the effects of LPS administration in M-CSF-deficient op/op mutant mice. The expression of cytokines and receptors in the liver was studied by immunohistochemistry and RT-PCR. Neutrophil infiltration in the liver was also examined. RESULTS: After LPS administration, cytokine production and expression of LPS receptors, such as CD14 and scavenger receptor class A (MSR-A), were induced at lower levels in op/op mice than those in littermate mice. Neutrophil infiltration in the liver of op/op mice did not differ significantly from that of littermate mice. Anti-IL-8 receptor homologue and anti-C5a receptor antibody reduced the number of infiltrating neutrophils. CONCLUSIONS: These findings indicate that deficient macrophage activation following LPS injection in op/op mice is associated with decreased expression of CD14 and MSR-A in the liver. Thus, M-CSF plays a critical role in LPS-induced macrophage activation but does not exert a dominant role in neutrophil infiltration in the liver.     

Stimulation of murine colony-forming cells with high proliferative potential by the combination of GM-CSF and CSF-1

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has previously been shown to stimulate granulocyte, macrophage, and megakaryocyte lineages to act as an erythroid burst-promoting activity and to stimulate limited replication of spleen colony-forming cells. Here we demonstrate that murine GM-CSF alone or in combination with macrophage colony-stimulating factor (CSF-1) can stimulate colony- forming cells in bone marrow (BM) that have a high proliferative capacity. In cultures of BM from mice treated with 5-fluorouracil (FU) eight days before sampling, GM-CSF alone or in combination with CSF-1 stimulated the formation of large macrophage colonies with diameters greater than 0.5 mm. CSF-1 alone, at 800 units or greater, also stimulated larger colonies; however, these colonies were always less than 1.1 mm in diameter, whereas GM-CSF in combination with CSF-1 stimulated many colonies with diameters between 1 and 4 mm. At all doses of CSF-1 tested, the combination of factors resulted in a synergistic increase in colonies with diameters greater than 1.0 or 2.0 mm. Analysis of the incidence of colony-forming cells in the BM of normal mice and mice 2, 4, 6, and 8 days after FU treatment demonstrated that the progenitor cells stimulated by GM-CSF alone or in combination with CSF-1 were depleted by FU treatment in vivo and regenerated more rapidly than did the macrophage progenitors (M-CFC) stimulated by CSF-1 alone. This is similar to the properties of the previously described high-proliferative potential, colony-forming cell (HPP-CFC) that is responsive to interleukin-3 plus CSF-1 but not the HPP-CFC stimulated by hematopoietin 1 plus CSF-1. These data suggest that GM-CSF plus CSF-1 act synergistically to stimulate a population of progenitor cells that have a high proliferative potential and have properties similar to those of the population of HPP-CFC stimulated by interleukin-3 plus CSF-1.     

Colony stimulating factor 1 is required for mammary gland development during pregnancy.

The study of colony stimulating factor 1 (CSF-1), a homodimeric serum growth factor that regulates mononuclear phagocytes and is involved in maternal-fetal interactions during pregnancy, was dramatically enhanced by the observation that the recessive mutation osteopetrosis, op, is an inactivating mutation in the CSF-1-encoding gene. Homozygous mutant (op/op) mice completely lack CSF-1, are osteopetrotic consequent to a deficiency in osteoclasts, have severely reduced numbers of macrophages, and have reduced fertility evident at the pre- and postimplantation stages of pregnancy. We show here that op/op females have a lactational defect, and consequently, although some are able to produce offspring, few nurture any pups and none feeds a full litter. This lactational defect is due to incomplete mammary gland ductal growth during pregnancy, a precocious development of the lobulo-alveolar system, and despite expression of milk proteins, a failure to switch to a lactational state. These data show that CSF-1 has a role in the development of the mammary gland during pregnancy.     

Efficient differentiation and function of human macrophages in humanized CSF-1 mice

Humanized mouse models are useful tools to understand pathophysiology and to develop therapies for human diseases. While significant progress has been made in generating immunocompromised mice with a human hematopoietic system, there are still several shortcomings, one of which is poor human myelopoiesis. Here, we report that human CSF-1 knockin mice show augmented frequencies and functions of human myeloid cells. Insertion of human CSF1 into the corresponding mouse locus of Balb/c Rag2(-/-) γc(-/-) mice through VELOCIGENE technology resulted in faithful expression of human CSF-1 in these mice both qualitatively and quantitatively. Intra-hepatic transfer of human fetal liver derived hematopoietic stem and progenitor cells (CD34(+)) in humanized CSF-1 (CSF1(h/h)) newborn mice resulted in more efficient differentiation and enhanced frequencies of human monocytes/macrophages in the bone marrow, spleens, peripheral blood, lungs, liver and peritoneal cavity. Human monocytes/macrophages obtained from the humanized CSF-1 mice show augmented functional properties including migration, phagocytosis, activation and responses to LPS. Thus, humanized mice engineered to express human cytokines will significantly help to overcome the current technical challenges in the field. In addition, humanized CSF-1 mice will be a valuable experimental model to study human myeloid cell biology.     

Slow receptor acquisition by NK cells regenerated in vivo from transplanted fetal liver or adult bone marrow stem cells

Adult mouse natural killer (NK) cells express two families of MHC class I-specific receptors, namely Ly49 and CD94/NKG2, whereas fetal and neonatal NK cells express only CD94/NKG2. After birth, Ly49(+) NK cells slowly increase and CD94/NKG2(+) NK cells decrease. The aim of this study was to determine whether murine NK cells develop differently from transplants of fetal liver and adult marrow stem cells and whether the adult marrow microenvironment is critical for NK receptor maturation. Enriched populations of stem cells were transplanted into adult mice, and the kinetics of NK receptor acquisition was examined. NK cells from osteopetrotic Csf1(op)/Csf1(op) mice, in which hematopoiesis within the marrow is severely limited, were also analyzed.NK cells regenerated from both fetal and adult stem cells initially resembled neonatal NK cells in their slow acquisition of Ly49 over several weeks, although the adult stem cell-derived NK cells matured approximately 10 days sooner. NK cells from adult Csf1(op)/Csf1(op) mice expressed normal levels of Ly49. Maturation of the NK receptor repertoire is a slow process regardless of their stem cell origin or reduced marrow space caused by osteopetrosis.     

Inducible production of human macrophage growth factor, CSF-1

A panel of human cell lines was screened for production of colony- stimulating factor-1 (CSF-1) using a specific radioreceptor assay and criterion of macrophage colony growth in mouse bone marrow culture. The pancreatic carcinoma lines MIA PaCa and PANC were found to secrete high levels of CSF-1. In a bone marrow proliferation assay, the activities from these two lines were blocked by a CSF-1 specific neutralizing antiserum, confirming the predominant content of this macrophage growth factor. MIA PaCA cells stopped secreting CSF-1 when transferred to various serum-free media. Serum-free production could be reinitiated by phorbol myristic acetate (PMA). Purified CSF-1 from serum-free MIA PaCa cells stimulated the formation of 14-day colonies from total and nonadherent mononuclear human bone marrow cells. Most of the colonies consisted exclusively of large, dispersed macrophages that were intensely stained for nonspecific esterase. Although similar numbers of human 14-day colonies were stimulated by CSF-1 and other CSFs, more CSF- 1 was required for the proliferation of human as compared with murine bone marrow progenitors. Northern analysis of mRNA from induced-MIA PaCa cells, using a human CSF-1 oligonucleotide probe, revealed multiple species of CSF-1 mRNA ranging from 1.5 to 4.5 kilobases (kb). Uninduced, serum-free cultures showed only the largest mRNA species, suggesting that serum removal interfered with CSF-1 mRNA processing related to synthesis and/or secretion of the protein. Regulation of the production of CSF-1 may be an important physiological process in hematopoiesis and macrophage functioning.