Delayed hematopoietic development in osteopetrotic (op/op) mice.

Changes in structure, cellularity, hematopoietic progenitor cell and macrophage content, and osteoclast activity were investigated in the hematopoietic organs of the colony-stimulating factor 1(CSF-1)-less osteopetrotic (op/op) mouse. The data indicated that op/op mice undergo an age-related hematopoietic recovery and resolution of osteopetrosis, suggesting that the hematopoietic system has the capacity to use alternative mechanisms to compensate for the absence of an important multifunctional growth factor, CSF-1. In young animals, op/op femurs were heavily infiltrated with bone, and marrow cellularity was significantly reduced. After 6 wk of age, there was an increase in the marrow space available for hematopoiesis. The femoral cavity of op/op mice progressively enlarged, and by 22 wk of age its appearance and marrow cellularity was comparable to that of controls. The percentage of op/op mononuclear phagocytes, defined by F4/80 antigen expression, progressively increased to normal levels by 35 wk of age. There was no difference in the incidence of both primitive and mononuclear phagocyte-committed, CSF-1-responsive progenitor cells in op/op marrow, but their femoral content was significantly reduced in young mice. During the period of reduced hematopoiesis in the marrow of young op/op mice, splenic hematopoietic activity was elevated. This mutant mouse represents a system for the study of the CSF-1-independent regulatory mechanisms involved in hematopoietic regulation.

Constitutive expression of exogenous myc in myelomonocytic cells: acquisition of a more transformed phenotype and inhibition of differentiation induction.

The effects of deregulated expression of the human c-myc and MC29 v-myc oncogenes have been examined in a murine myelomonocytic cell line J774 (c-myc) and in a variety of myelomonocytic cell lines of different degrees of maturity generated from primary hematopoietic tissue (v-myc). Introduction of a Moloney murine leukemia virus long terminal repeat (LTR) c-myc construct into J774 cells resulted in constitutive expression of the exogenous myc gene and a concomitant increase in the degree of transformation and tumorigenicity of the cells. In addition, constitutive expression of exogenous myc inhibited induced differentiation of these cells by a variety of treatments including addition to the medium of lipopolysaccharide (LPS) or the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) as well as complete withdrawal of serum from the medium. The degree of increased transformation, tumorigenicity and inhibition of terminal differentiation was dependent upon the level of exogenous myc expression. For the v-myc-generated myelomonocytic cell lines, introduction of v-myc resulted in a high degree of transformation and, irrespective of the differentiation status of the cells, a block of induced differentiation. These results indicate that the level of constitutive myc expression can affect the transformed phenotype, tumorigenicity and differentiation inducibility of myelomonocytic cells.

Inducible transformation of fibroblasts using a metallothionein-v-myc gene construct.

An inducible oncogene construct has been engineered by coupling the MC29 v-myc oncogene to the sheep metallothionein promoter. Transfection of this plasmid, which also contains the neomycin resistance gene, into Rat-1 cells, has resulted in the isolation of independent clones resistant to G418. Certain of these clones were found to exhibit inducible transformation in response to ZnSO4. Transformation was graded with increasing ZnSO4 levels and was reversible when ZnSO4 was removed from the media. By analyzing v-myc mRNA levels, the inducible alterations in cellular morphology and growth were found to be associated with increased v-myc expression. The metallothionein promoter exhibited negligible constitutive expression of v-myc and none of the clones isolated exhibited spontaneous transformation. Our results show that the use of a metallothionein promoter v-myc construct facilitates the study of inducible fibroblast transformation.

An in vitro fibroblast model system to study myc-driven tumour progression.

We have utilized fibroblast cell lines to investigate myc-mediated cell transformation and tumour progression. Deregulated myc alleles were introduced into the rat fibroblast cell line, Rat-1, and a partially-transformed derivative of this cell line termed Rat-1 (PT). A human c-myc gene coupled to the Moloney murine leukemia virus long terminal repeat was introduced into both cell lines by transfection. The avian MC29 v-myc gene was introduced into the Rat-1 cell line by retroviral infection using a Moloney murine leukemia recombinant retrovirus. For both cell lines, the introduction of exogenous myc genes resulted in an increased degree of transformation. For the non-tumorigenic Rat-1 cell line, this also resulted in the acquisition of tumorigenicity, while for the Rat-1 (PT) cell line the degree of tumorigenicity was increased. Various clones were isolated and, for both human c-myc and avian v-myc, the level of myc expression correlated with the degree of transformation and the tumorigenic potential of the cell lines. In addition, both these parameters could be increased by passaging through syngeneic recipients. Our data show that tumour progression may be driven by the deregulated expression of myc genes; that transformation and tumorigenicity correlate with the level of exogenous myc expression; that additional events involving both in vitro and in vivo selection are involved in this process; and that myc expression may increase the cells' metastatic capacity.

Absence of colony-stimulating factor-1 in osteopetrotic (csfmop/csfmop) mice results in male fertility defects.

Previous studies have shown that the mononuclear phagocyte growth factor, colony-stimulating factor-1 (CSF-1), has an important role in female reproduction. Mating experiments with osteopetrotic (csfmop/csfmop) mice, which possess an inactivating mutation in the CSF-1 gene, suggested that there are male, as well as female, reproductive defects. In the present study, we have shown that male csfmop/csfmop mice have a sevenfold lower concentration of circulating testosterone (T) and a significantly lower intratesticular T concentration than wild-type mice. These lowered T concentrations were associated with a reduction in mating capability and a reduction in the number of viable sperm. Reconstitution of male csfmop/csfmop mice with either circulating T in the adult or circulating CSF-1 throughout the postnatal period completely restored viable sperm numbers and significantly restored sexual behavior. These observations, coupled with the close association of Leydig cells with testicular macrophages and the proposed function of these macrophages in the regulation of Leydig cell steroidogenesis, suggest that CSF-1-regulated testicular macrophages play an important role in male reproduction.

Rapid differentiation of Australian, European and American ranaviruses based on variation in major capsid protein gene sequence.

Epizootic haematopoietic necrosis virus (EHNV), Bohle iridovirus (BIV) and Wamena virus (WV) cause serious diseases in fish, amphibians and snakes, respectively but are restricted to Australasia. European catfish virus (ECV) and sheatfish virus (ESV) have caused epizootics in fish on farms in continental Europe. Currently there are no simple or readily available methods to distinguish these viruses, which are in the Iridoviridae. They are culturally, morphologically and antigenically very similar to Frog Virus 3 (FV3), the type species in Ranavirus in this family and Gutapo virus (GV), another amphibian ranavirus from America. The diseases caused by EHNV, ESV and ECV are so serious that they are internationally notifiable. Tests to distinguish these viruses are desirable to ensure that disease occurrences do not unnecessarily restrict trade in aquaculture products. The gene encoding the major capsid protein from two EHNV isolates from different fish species (Perca fluviatilis and Oncorhynchus mykiss) and one BIV isolate were sequenced and the data and deduced amino acid sequences were compared with those from FV3 and other iridoviruses. The sequences for the two EHNV isolates were identical, confirming suggestions from existing partial MCP sequence that the same type of EHNV infects wild redfin perch and farmed rainbow trout. Differences in restriction endonuclease patterns of specific PCR products were predicted and confirmed between EHNV, BIV, and WV and provided a basis for rapid differentiation of these viruses from each other and from ESV/ECV and FV3/GV. These simple and rapid tests to distinguish important ranaviruses from the regions of Europe, Australia and America will help regulatory authorities assess the need for disease control responses in the event of occurrence of ranavirus infection in aquaculture species.

Osteopetrotic (op/op) mice deficient in macrophages have the ability to mount a normal T-cell-dependent immune response.

The osteopetrotic (op/op) mouse possesses an inactivating mutation in the CSF-1 gene that is associated with a lack of certain mononuclear phagocyte populations. To examine the effects of these deficiencies on T-cell-dependent immune functions, the responses of op/op and normal mice to a T-cell-dependent antigen, ovalbumin, and to a foreign histocompatibility antigen were compared. The ability of op/op mice (1) to induce antigen-specific proliferation of naive T-cells, (2) to generate cytotoxic T-lymphocytes, (3) to supply spleen cells to serve as stimulators in a mixed lymphocyte reaction, and (4) to produce IgM and IgG antibodies was indistinguishable from normal mice. These data are consistent with the involvement of specific macrophages, or other cells whose development is independent of CSF-1, in these immune responses.

hFOG-2, a novel zinc finger protein, binds the co-repressor mCtBP2 and modulates GATA-mediated activation.

We have identified a novel human zinc finger protein, hFOG-2, which is related to but distinct from the murine transcription factor Friend-of-GATA-1 (mFOG-1). The hFOG-2 gene was initially detected in K562 cells using a polymerase chain reaction approach with degenerate primers corresponding to zinc finger regions of mFOG-1. A murine homologue of hFOG-2 was also identified in the mouse expressed sequence tag data banks, indicating that a family of FOG genes exists in mammals. hFOG-2 appears to be widely expressed, while mFOG-1 is expressed primarily in erythroid and megakaryocytic cells and plays a fundamental role in the development of these lineages. Sequencing of the full-length hFOG-2 cDNA indicates that the interaction domains for transcription factors GATA-1 and mCtBP2 are both conserved and we have shown that this new FOG protein also physically interacts with these factors. We have demonstrated that hFOG-2, like mFOG-1, can act in concert with GATA-1 to activate gene expression from the p45 NF-E2 promoter region, but that it can also act to repress GATA-mediated activation of additional reporter constructs. Finally, we have identified a repression domain in hFOG-2 and show that repression is dependent upon the integrity of the mCtBP2 interaction motif Pro-Ile-Asp-Leu-Ser.

The role of platelet alpha-granular proteins in the regulation of thrombopoietin messenger RNA expression in human bone marrow stromal cells.

Thrombopoietin (TPO), the specific cytokine that regulates platelet production, is expressed in human bone marrow (BM), kidney, and liver. There appears to be no regulation of TPO in the kidney and liver, but TPO messenger RNA (mRNA) expression can be modulated in the stromal cells of the BM. In this study, we used primary human BM stromal cells as a model to study the regulation of TPO mRNA expression in response to various platelet alpha-granular proteins. We showed that platelet-derived growth factor (PDGF) BB and fibroblast growth factor (FGF) 2 stimulated TPO mRNA expression in both a dose-dependent and time-dependent manner. The addition of 50 ng/mL of PDGF and 20 ng/mL of FGF resulted in maximal induction of TPO mRNA expression in 4 hours. We also found that platelet factor 4 (PF4), thrombospondin (TSP), and transforming growth factor-beta (TGF-beta) are negative modulators of megakaryocytopoiesis. We observed suppression in TPO mRNA expression with 1 microg/mL of both PF4 and TSP and 50 ng/mL of TGF-beta, with maximal suppression occurring 4 hours after the addition of these proteins. Finally, the addition of whole-platelet lysate produced a dose-dependent inhibition of TPO expression. On the basis of these findings, we propose that the platelet alpha-granular proteins studied may regulate TPO gene expression in BM stromal cells by means of a feedback mechanism.

Role of colony stimulating factor-1 in the establishment and regulation of tissue macrophages during postnatal development of the mouse.

Colony stimulating factor-1 (CSF-1) regulates the survival, proliferation and differentiation of mononuclear phagocytes. The osteopetrotic (op/op) mutant mouse is devoid of CSF-1 due to an inactivating mutation in the CSF-1 gene and is deficient in several mononuclear phagocyte subpopulations. To analyze more fully the requirement for CSF-1 in the establishment and maintenance of mononuclear phagocytes, the postnatal development of cells bearing the macrophage marker antigens F4/80 and MOMA-1, in op/op mice and their normal (+/op or +/+) littermates, were studied during the first three months of life. In normal mice, maximum expression of tissue F4/80+ cells was generally correlated with the period of maximum organogenesis and/or cell turnover. Depending on the tissue, the F4/80+ cell density either decreased, transiently increased or gradually increased with age. In op/op mice, tissues that normally contain F4/80+ cells could be classified into those in which F4/80+ cells were absent and those in which the F4/80+ cell densities were either reduced, normal or initially normal then subsequently reduced. To assess which F4/80+ populations were regulated by circulating CSF-1 in normal mice, op/op mice in which the circulating CSF-1 concentration was restored to above normal levels by daily subcutaneous injection of human recombinant CSF-1 from day 3 were analyzed. These studies suggest that circulating CSF-1 exclusively regulates both the F4/80+ cells in the liver, spleen and kidney and the MOMA-1+ metallophilic macrophages in the spleen. Macrophages of the dermis, bladder, bone marrow and salivary gland, together with a subpopulation in the gut, were partially restored by circulating CSF-1, whereas macrophages of the muscle, tendon, periosteum, synovial membrane, adrenals and the macrophages intimately associated with the epithelia of the digestive tract, were not corrected by restoration of circulating CSF-1, suggesting that they are exclusively locally regulated by this growth factor. Langerhans cells, bone marrow monocytes and macrophages of the thymus and lymph nodes were not significantly affected by circulating CSF-1 nor decreased in op/op mice, consistent with their regulation by other growth factors. These results indicate that important differences exist among mononuclear phagocytes in their dependency on CSF-1 and the way in which CSF-1 is presented to them. They also suggest that the prevalent role of CSF-1 is to influence organogenesis and tissue turnover by stimulating the production of tissue macrophages with local trophic and/or scavenger (physiological) functions. Macrophages involved in inflammatory and immune (pathological) responses appear to be dependent on other factors for their ontogenesis and function.(ABSTRACT TRUNCATED AT 400 WORDS)

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.