Absence Of CSF-1 Research Articles

CSF-1-activated macrophages are target-directed and essential mediators of Schwann cell dedifferentiation and dysfunction in Cx32-deficient mice.

We investigated connexin 32 (Cx32)-deficient mice, a model for the X-linked form of Charcot-Marie-Tooth neuropathy (CMT1X), regarding the impact of low-grade inflammation on Schwann cell phenotype. Whereas we previously identified macrophages as amplifiers of the neuropathy, we now explicitly focus on the impact of the phagocytes on Schwann cell dedifferentiation, a so far not-yet addressed disease-related mechanism for CMT1X. Using mice heterozygously deficient for Cx32 and displaying both Cx32-positive and -negative Schwann cells in one and the same nerve, we could demonstrate that macrophage clusters rather than single macrophages precisely associate with mutant but not with Cx32-positive Schwann cells. Similarly, in an advanced stage of Schwann cell perturbation, macrophage clusters were strongly associated with NCAM- and L1-positive, dedifferentiated Schwann cells. To clarify the role of macrophages regarding Schwann cell dedifferentiation, we generated Cx32-deficient mice additionally deficient for the macrophage-directed cytokine colony-stimulating factor (CSF)-1. In the absence of CSF-1, Cx32-deficient Schwann cells not only showed the expected amelioration in myelin preservation but also failed to upregulate the Schwann cell dedifferentiation markers NCAM and L1. Another novel and unexpected finding in the double mutants was the retained activation of ERK signaling, a pathway which is detrimental for Schwann cell homeostasis in myelin mutant models. Our findings demonstrate that increased ERK signaling can be compatible with the maintenance of Schwann cell differentiation and homeostasis in vivo and identifies CSF-1-activated macrophages as crucial mediators of detrimental Schwann cell dedifferentiation in Cx32-deficient mice.

Production and characterisation of a monoclonal antibody that recognises the chicken CSF1 receptor and confirms that expression is restricted to macrophage-lineage cells

Macrophages contribute to innate and acquired immunity as well as many aspects of homeostasis and development. Studies of macrophage biology and function in birds have been hampered by a lack of definitive cell surface markers. As in mammals, avian macrophages proliferate and differentiate in response to CSF1 and IL34, acting through the shared receptor, CSF1R. CSF1R mRNA expression in the chicken is restricted to macrophages and their progenitors. To expedite studies of avian macrophage biology, we produced an avian CSF1R-Fc chimeric protein and generated a monoclonal antibody (designated ROS-AV170) against the chicken CSF1R using the chimeric protein as immunogen. Specific binding of ROS-AV170 to CSF1R was confirmed by FACS, ELISA and immunohistochemistry on tissue sections. CSF1 down-regulated cell surface expression of the CSF1R detected with ROS-AV170, but the antibody did not block CSF1 signalling. Expression of CSF1R was detected on the surface of bone marrow progenitors only after culture in the absence of CSF1, and was induced during macrophage differentiation. Constitutive surface expression of CSF1R distinguished monocytes from other myeloid cells, including heterophils and thrombocytes. This antibody will therefore be of considerable utility for the study of chicken macrophage biology.

Absence of colony stimulation factor-1 receptor results in loss of microglia, disrupted brain development and olfactory deficits.

The brain contains numerous mononuclear phagocytes called microglia. These cells express the transmembrane tyrosine kinase receptor for the macrophage growth factor colony stimulating factor-1 (CSF-1R). Using a CSF-1R-GFP reporter mouse strain combined with lineage defining antibody staining we show in the postnatal mouse brain that CSF-1R is expressed only in microglia and not neurons, astrocytes or glial cells. To study CSF-1R function we used mice homozygous for a null mutation in the Csflr gene. In these mice microglia are >99% depleted at embryonic day 16 and day 1 post-partum brain. At three weeks of age this microglial depletion continues in most regions of the brain although some contain clusters of rounded microglia. Despite the loss of microglia, embryonic brain development appears normal but during the post-natal period the brain architecture becomes perturbed with enlarged ventricles and regionally compressed parenchyma, phenotypes most prominent in the olfactory bulb and cortex. In the cortex there is increased neuronal density, elevated numbers of astrocytes but reduced numbers of oligodendrocytes. Csf1r nulls rarely survive to adulthood and therefore to study the role of CSF-1R in olfaction we used the viable null mutants in the Csf1 (Csf1op) gene that encodes one of the two known CSF-1R ligands. Food-finding experiments indicate that olfactory capacity is significantly impaired in the absence of CSF-1. CSF-1R is therefore required for the development of microglia, for a fully functional olfactory system and the maintenance of normal brain structure.

Inappropriate Expression of CSF-1 in CSF-1R-Expressing Cells in Mice Leads to Osteoporosis, Macrophage Activation and Early Death.

The CSF-1R is selectively expressed on mononuclear phagocytes, osteoclasts and cells of the female reproductive tract. CSF-1 is normally expressed in a wide variety of cell types, excluding those expressing the CSF-1 receptor (CSF-1R). Autocrine/paracrine regulation by CSF-1 plays an important role in the development of disease in mouse models of acute myeloid leukemia and mammary tumor progression to metastasis. To determine the consequences of autocrine regulation by CSF-1 in the absence of other interventions, we generated transgenic mice that expressed CSF-1 in CSF-1R bearing cells. In contrast to mice that expressed the CSF-1R in CSF-1-expressing cells, which had no gross phenotype at 1 year of age, mice expressing CSF-1 under the control of mouse CSF-1R promoter and second intron (TgRC mice), had a short lifetime, starting to lose weight and mobility after 3 weeks of age. CSF-1 mRNA was expressed in peritoneal macrophages (PM) and sequence analysis of the RT-PCR products verified the expression of a chimeric mRNA of the CSF-1R exon 2, 3 and CSF-1 mRNAs under control of the CSF-1R promoter. Quantitative real-time RT-PCR analysis revealed that CSF-1 mRNA expression was increased by 19-fold in PM and 980-fold in bone marrow derived macrophages (BMM) from a TgRC founder mouse, compared to these populations from non-transgenic mice. Consistent with autocrine regulation by CSF-1, in contrast to BMM from non-transgenic mice, TgRC BMM proliferated in the absence of CSF-1. However, there was no elevation of the circulating CSF-1 in TgRC mice, indicating that transgene-regulated expression of CSF-1 only affected local CSF-1 concentrations. X-ray and histological analysis demonstrated that TgRC mice developed osteoporosis with increased numbers of femoral TRAP-positive osteoclasts. Macrophage densities were significantly increased in bone marrow, liver, spleen and brain. In several tissues, there were local regions of increased macrophage density, consistent with effects of locally produced CSF-1 on macrophage survival and/or proliferation. Also, macrophages in several tissues had a more dendritic appearance than in non-transgenic mice, suggesting that they may be activated. TgRC PM, cultured in the presence of CSF-1 exhibited a highly dendritic morphology with multiple protruding pseudopods also suggesting an activated state. Studies of the macrophage cytokine expression profile by the real-time quantitative RT-PCR revealed elevated levels of mRNA for the proinflammatory cytokines, IL-1β (33-fold) (in BMM) and IL-10 (10-fold) and IL-6 (2.6-fold) (in PM), consistent with the priming effect of CSF-1 on proinflammatory cytokine release by macrophages. These studies indicate that inappropriate local expression of CSF-1 by or near CSF-1R-expressing cells can result in activation of macrophages and osteoclasts and severe disease. Supported by NIH grant CA32551 (ERS) and an ASH Fellow Scholar Award and Leukemia and Lymphoma Society Special Fellow Award (X-MD).

Phosphorothioate Backbone Modification Modulates Macrophage Activation by CpG DNA

Macrophages respond to unmethylated CpG motifs present in nonmammalian DNA. Stabilized phosphorothioate-modified oligodeoxynucleotides (PS-ODN) containing CpG motifs form the basis of immunotherapeutic agents. In this study, we show that PS-ODN do not perfectly mimic native DNA in activation of macrophages. CpG-containing PS-ODN were active at 10- to 100-fold lower concentrations than corresponding phosphodiester ODN in maintenance of cell viability in the absence of CSF-1, in induction of NO production, and in activation of the IL-12 promoter. These enhancing effects are attributable to both increased stability and rate of uptake of the PS-ODN. By contrast, PS-ODN were almost inactive in down-modulation of the CSF-1R from primary macrophages and activation of the HIV-1 LTR. Delayed or poor activation of signaling components may contribute to this, as PS-ODN were slower and less effective at inducing phosphorylation of the extracellular signal-related kinases 1 and 2. In addition, at high concentrations, non-CpG PS-ODN specifically inhibited responses to CpG DNA, whereas nonstimulatory phosphodiester ODN had no such effect. Although nonstimulatory PS-ODN caused some inhibition of ODN uptake, this did not adequately explain the levels of inhibition of activity. The results demonstrate that the phosphorothioate backbone has both enhancing and inhibitory effects on macrophage responses to CpG DNA.

CSF-1 (M-CSF) Enhances the Inflammatory Response of Fibronectin-Primed Macrophages: Pathways Involved in Activation of the Cytokine Network

We have previously reported that the priming of thioglycollate-elicited peritoneal macrophages (PMφ), as a representative population of mononuclear phagocytes (MNP), by macrophage-colony-stimulating factor (M-CSF or CSF-1) rendered these cells more susceptible to secondary stimulation by extracellular matrix (ECM) proteins, in particular fibronectin (FN), and that at least two β1 integrins, VLA 4 (α₄β₁ or CD49d) and VLA 5 (α₅β₁ or CD49e), regulate IL-6 gene expression when PMφ come into contact with FN. In this report, we focused our attention on resident PMφ, as a more mature/differentiated MNP subpopulation. By using granulocyte-macrophage colony-stimulating factor (GM-CSF)- and IL-6-knockout (null) mice, we demonstrated that the cooperative effect between CSF-1 and FN in IL-6 release was a result of a sequential stimulation of the GM-CSF, but not the TNF-α, gene via interaction with VLA 5. We also showed that regardless of the presence or absence of CSF-1 or FN, IL-6 inhibits GM-CSF and TNF-α gene expression in an autocrine manner. The observed effects were specific because CSF-1 enhanced VLA 5 expression and blocking FN-treated resident PMφ in vitro with VLA 5 monoclonal antibodies inhibited the IL-6 response. We found that treatment of resident PMφ with the protein kinase C inhibitor, staurosporine, and the activator, phorbol myristate acetate (PMA), resulted in marked modulation of either FN- or FN/CSF-1-induced cytokine release. An increased level of VLA 5 expression was observed in PMA-treated resident PMφ. We concluded that in inflammatory processes, CSF-1 drives a number of pathways involved in the regulation of the expression of several genes and renders MNP highly susceptible to stimulation by ECM proteins that transform the MNP into secretory inflammatory cells.

Postnatal mammary gland development requires macrophages and eosinophils.

Interactions between mammary epithelial and mesenchymal cells including fibroblasts and adipocytes are crucial for the proper postnatal development of the mammary ductal tree. Often overlooked, however, are the migrant cells that enter tissues at different stages of development. In this paper we identify two such cell types, macrophages and eosinophils, that are recruited around the growing terminal end buds (TEBs) during postnatal development. An important role for leukocytes in mammary gland ductal outgrowth is first demonstrated by depleting mice of leukocytes using sub-lethal (gamma)-irradiation. This treatment results in a curtailment of mammary gland epithelial development that is completely rescued by bone-marrow transplantation, concurrent with a restoration of macrophage and eosinophil recruitment around the growing ducts. Using mice homozygous for a null mutation in the gene for CSF1 (Csfm(op)/Csfm(op)), the major growth factor for macrophages, we show that in the absence of CSF1, the population of macrophages in mammary glands is depleted. In this mutant, the formation of TEBs, their outgrowth into the fat pad and the branching of the resultant ducts are all impaired. Similarly, by using mice homozygous for a null mutation in the gene for eotaxin, a major chemokine for local recruitment of eosinophils in tissue, we identify eotaxin as the necessary and sufficient chemokine responsible for eosinophil recruitment around TEBs. In the absence of eosinophils, mammary gland branch formation and to a lesser extent TEB formation are reduced. Our data show that CSF1-regulated macrophages, in collaboration with eotaxin-regulated eosinophils, have essential and complementary functions in regulating the branching morphogenesis of the mammary gland.

MRL/lprand MRL+/+ Macrophage DNA Synthesis in the Absence and the Presence of Colony-Stimulating Factor-1 and Granulocyte-Macrophage Colony-Stimulating Factor

AbstractMacrophage accumulation and proliferation as well as altered macrophage properties have been observed in autoimmune MRL mice. To determine whether there might be innate differences in the proliferative responses, we examined the DNA synthesis responses of peritoneal macrophages and macrophages derived in vitro from bone marrow precursors (bone marrow-derived macrophages (BMM)). Murine peritoneal exudate macrophages normally require the addition of macrophage CSF (CSF-1) to enter cell cycle in vitro. In contrast, we have found that many thioglycollate-induced adherent peritoneal macrophages, but not resident peritoneal macrophages, from both MRL/lpr and MRL+/+ mice atypically underwent DNA synthesis even in the absence of added CSF-1. They also responded very well to granulocyte-macrophage CSF. These findings may help to explain the appearance of increased macrophage numbers in MRL lesions. In contrast to a previous report, it was found that MRL/lpr and MRL+/+ BMM did not have an enhanced response to CSF-1 and that modulation of CSF-1 receptor expression was not more rapid in MRL BMM. We also found no evidence for abnormal CSF-1 internalization and degradation or for the lpr mutation to have any enhanced effect on BMM survival in the absence of CSF-1. TNF-α lowered the DNA synthesis response to CSF-1 of MRL/lpr BMM rather than enhanced it, as has been reported. Our data suggest that the enhanced accumulation of macrophages in the MRL/lpr kidney cannot be explained by a proposed model of enhanced responsiveness of MRL/lpr BMM to CSF-1, including a contribution by TNF-α.

Oligomerization of the Fes tyrosine kinase. Evidence for a coiled-coil domain in the unique N-terminal region.

The c-fes proto-oncogene encodes a non-receptor tyrosine kinase (Fes) that has been implicated in cytokine receptor signal transduction and myeloid differentiation. Previous work from our laboratory has shown that Fes autophosphorylates via an intermolecular mechanism more commonly associated with growth factor receptor tyrosine kinases. Analysis of the Fes amino acid sequence with the COILS algorithm indicates that the N-terminal region of the protein has a very high probability of forming coiled-coil structures often associated with oligomeric proteins. These findings suggest that oligomerization may be a prerequisite for trans-autophosphorylation and activation of Fes. To establish whether the active form of Fes is oligomeric, we performed gel-filtration experiments with recombinant Fes and found that it eluted as a single symmetrical peak of approximately 500 kDa. No evidence of the monomeric, 93-kDa form of the protein was observed. Deletion of the unique N-terminal domain (amino acids 1-450, including the coiled-coil homology region) completely abolished the formation of oligomers. Furthermore, co-precipitation assays demonstrated that an immobilized glutathione S-transferase fusion protein containing the Fes N-terminal region bound to full-length Fes but not to a mutant lacking the N-terminal region. Similarly, a recombinant Fes N-terminal domain protein was readily cross-linked in vitro, whereas the SH2 and kinase domains were refractory to cross-linking. Incubation of wild-type Fes with a kinase-inactive Fes mutant or with the isolated N-terminal region suppressed Fes autophosphorylation in vitro, suggesting that oligomerization may be essential for autophosphorylation of full-length Fes. The presence of an oligomerization function in the Fes family of tyrosine kinases suggests a novel mechanism for non-receptor protein-tyrosine kinase regulation.

Phosphatidylcholine signaling in response to CSF-1.

The formation of cell membrane following CSF-1 stimulation of a macrophage cell line is coordinated with cell cycle progression. The majority of membrane phospholipid accumulates during the S phase and results from cell-cycle dependent oscillations in the rates of phosphatidylcholine biosynthesis and degradation. Both synthesis and degradation are enhanced during the G1 phase, resulting in a high rate of phosphatidylcholine turnover. Degradation of phosphatidylcholine after CSF-1 stimulation is mediated by a phospholipase C, and the release of diacylglycerol during G1 phase is biphasic. The degradation essentially stops during the S phase, thus allowing biosynthesis to supply the necessary membrane for cell division and doubling. The degradation of phosphatidylcholine during G1 signals the downstream activation of c-fos and junB transcription and can be mimicked by incubation of the macrophage cells with exogenous bacterial phospholipase C. In contrast, the expression of c-myc transcripts normally associated with CSF-1 stimulation is severely compromised in phospholipase C-treated cells, indicating that the diacylglycerol signals a pathway distinct from the pathway that governs c-myc activation. Constitutive expression of c-myc complements phospholipase C activity and permits the growth of cells in the presence of exogenous bacterial enzyme and the absence of CSF-1. Protein kinase C is not required to mediate the diacylglycerol signal that supports cell growth. GTP exchange on Ras is not enhanced, and MAP kinase activity is not stimulated in response to phosphatidylcholine degradation by exogenous phospholipase C. The 85 kDa cytoplasmic phospholipase A2 is activated, however, as well as a novel protein we have called p96. Rapid serine phosphorylation of p96 follows stimulation of cells with either CSF-1 or exogenous phospholipase C. Analysis of the murine cDNA encoding p96 reveals an amino-terminal domain with significant similarity to the amino-terminal domain of the Drosophila-disabled gene product and a carboxy-terminal domain containing proline-rich sequences characteristic of SH3 binding regions. The sequence of p96 suggests an interactive role for this unique protein in the CSF-1 signal transduction cascade.

Regulation of microglial activation by TGF-beta, IL-10, and CSF-1.

Microglia are the resident macrophages of the brain and as such are active participants in immune responses in the central nervous system. Normal resting microglia express low levels of MHC class I and class II antigens and do not produce proinflammatory cytokines. However, microglial immune functions are induced in areas of infection or injury. To understand regulation of cytokines that are secreted by and act upon microglia, we examined production of interleukin (IL) -12, tumor necrosis factor-alpha (TNF-alpha), and nitric oxide (NO) by lipopolysaccharide (LPS)-stimulated microglia. We observed secretion of IL-12, TNF-alpha, and NO following stimulation of microglia with LPS. Addition of IL-10 suppressed TNF-alpha, IL-12, and NO production. Transforming growth factor-beta (TGF-beta) also inhibited TNF-alpha and NO but did not affect IL-12 secretion. IL-12 secretion became sensitive to TGF-beta inhibition when microglia were cultured in the absence of CSF-1. In addition to its effect on the response to TGF-beta, CSF-1 suppressed the response of microglia to LPS. These data suggest that CSF-1 may contribute to the immunologically privileged status of the central nervous system.

CSF-1 deficiency in mice results in abnormal brain development.

Colony stimulating factor-1 (CSF-1) was initially identified as a growth factor for mononuclear phagocytes. This study examines the role of CSF-1 in the development of the central nervous system (CNS). CSF-1 treatment of neurons cultured from embryonic brain promoted survival and process outgrowth in a dose-dependent manner. By contrast, CSF-1 treatment of neurons cultured from the osteopetrotic (op/op) mouse, a null mutant for CSF-1, promoted significantly less process outgrowth, suggesting that there are neural abnormalities in op/op animals. Nuclease protection assays were used to determine whether CSF-1 and its receptor are expressed at times appropriate to regulate neural development. Both CSF-1 and its receptor are expressed in developing mouse brain, with a unique pattern of CSF-1 mRNA splice variant expression encoding secreted, and not membrane-bound, growth factor. To determine whether brain function is altered by null mutation of CSF-1, op/op mice were examined using electrophysiologic assays. Brainstem auditory and visual evoked potentials were both abnormal in op/op mice. Further, intracortical recordings revealed aberrant neuronal function within visual cortex and alterations in the cortical circuitry that balances excitation and inhibition. Daily CSF-1 injection of postnatal op/op mice largely rescued the abnormal neural phenotype, confirming that the absence of CSF-1 during development is responsible for the abnormalities. The effects of CSF-1 on cultured embryonic neural cells, the developmentally appropriate expression of CSF-1 and its receptor, and the neurological abnormalities in op/op mice suggest a role for CSF-1 in brain development.

The development and establishment of hemopoiesis in fetal and newborn osteopetrotic ( op/op) mice

Previous studies of young CSF-1-less osteopetrotic (op/op) mice demonstrate a severe deficiency of both macrophages and osteoclasts, resulting in excessive bone formation, occlusion of the marrow cavity, and reduced hemopoietic activity. The accompanying splenomegaly and prolonged splenic hemopoiesis observed in these mice suggests that osteopetrosis may perturb the normal progression of fetal hemopoietic development and obstruct the seeding of hemopoietic precursors into the bone marrow. This study demonstrates that the absence of CSF-1 does not affect the progression of hemopoietic development in fetal op/op mice until after colonization of the bone marrow. Significant deficiencies in marrow cellularity and progenitor cell content in the long bones of op/op mice were not evident prior to Day 2 postnatal, suggesting that the altered hemopoietic state of young op/op mice is not a consequence of abnormal fetal hemopoietic development, but is primarily due to the lack of functional osteoclasts in op/op fetuses and hence, impaired remodeling of the marrow cavity after birth.

Transduction of a signal for arachidonic acid metabolism by untriggered CSF-1 receptor induces an opposite effect to that induced by CSF-1 receptor and its ligand: Separate regulation of phospholipase A 2 and cyclooxygenase by CSF-1 receptor/CSF-1

The mouse hematopoietic cell line, 32D, was transfected with c-fms, which encodes for the CSF-1 receptor, a tyrosine kinase (TK). In the absence of CSF-1, transfected cells show moderate levels of arachidonic acid (AA) release and produce a substantial amount of prostaglandin E 2 (PGE 2) in comparison with the original cell line. Exposure of transfected cells to CSF-1, while inducing a substantial increase in arachidonate release, nevertheless resulted in inhibition of PGE 2 production. Addition of ST638, a tyrosine kinase inhibitor, to cells transfected with c-fms in the absence of CSF-1 inhibited PGE 2 production within 10–60 min. Its addition to the same cells in the presence of CSF-1 induced an opposite effect, but required longer treatment (24 h). In either cell type, AA release was not affected by this agent. These data indicate that CSF-1 may regulate cyclooxygenase activity. The different effect of CSF-1 receptor on PGE 2 production in the presence or absence of CSF-1 and the opposite effect of a tyrosine kinase inhibitor on PGE 2 suggest that both the receptor alone or the receptor-ligand complex may transduce an active, but different, signal through tyrosine phosphorylation. CSF-1 receptor and CSF-1 may exert separate, but related, effects on phospholipase A 2 and cyclooxygenase activity which, in concert, or along with other tyrosine kinases, regulate prostaglandin production.

Alterations in CSF-1 Receptor Expression and Protein Tyrosine Phosphorylation in Autonomous Mutants of a CSF-1 Dependent Macrophage Cell Line

One approach to the problem of growth factor signal transduction is to identify alterations in growth factor-regulated events in mutants possessing an altered proliferative response. This approach was used in a study of the alterations in protein tyrosine phosphorylation in 22 independently-arising autonomous mutants of the CSF-1-dependent mouse macrophage cell line, BAC1.2F5. Only 4 of the mutants produced CSF-1 and/or factors that were capable of down-regulating the CSF-1 receptor (CSF-1R), suggesting that the majority were altered in the signal transduction pathway for proliferation. All of the mutants possessed lower numbers (13-89%) of cell surface CSF-1Rs than wild type cells. With two possible exceptions, the phosphorylation of the anti-phosphotyrosine-reactive cell surface CSF-1Rs from CSF-1 stimulated cells was directly proportional to cell surface CSF-1R number. In the absence of CSF-1, three of the 22 mutants exhibited tyrosine phosphorylation of proteins that was not observed in wild type cells under the same conditions. The results indicate that this approach will be useful in the analysis of the growth factor regulated pathway for cell proliferation.

Functional Expression of the Human Receptor for Colony-Stimulating Factor 1 (CSF-1) in Hamster Fibroblasts: CSF-1 Stimulates Na+/H+Exchange and DNA-Synthesis in the Absence of Phosphoinositide Breakdown

The human CSF-1 receptor (c-fms protooncogene product) was introduced into CSF-1-unresponsive Chinese hamster lung fibroblasts (CCL39 cell line) in order to study its coupling to biochemical signal-transducing systems and to compare the growth-regulating properties of CSF-1 to those of other growth factors. Independent clones expressing different levels of CSF-1 receptors were isolated and characterized. CSF-1 increased [3H]thymidine incorporation in serum-starved cells and potentiated the mitogenic effects of FGF and thrombin. As already observed for other growth factors activating receptor tyrosine kinases (EGF, FGF, IGF-I), CSF-1 alone did not trigger inositol phosphate formation, but slightly enhanced the activity of phospholipase C agonists (thrombin, A1F4- complex). Activation of the CSF-1 receptor by its ligand was evidenced by the rapid activation of the Na+/H+ exchanger resulting in amiloride-sensitive cytoplasmic alkalinization (0.1-0.2 pH units) within minutes after stimulation. Whereas pertussis toxin does not affect the action of EGF, FGF, or IGF-I in CCL39 cells, it partially inhibited both DNA synthesis reinitiation and activation of Na+/H+ exchange by CSF-1, indicating that the CSF-1 receptor can communicate with a signal-transducing GTP binding protein. A point-mutated form of the c-fms gene product, in which Tyr 969, a residue negatively modulating signal transduction, had been replaced with Phe [fms (F969)], did not generate responses significantly different from those obtained with the wild-type c-fms gene product. In the absence of CSF-1, cells expressing either wild-type or fms (F969) showed a considerably higher basal level of thymidine incorporation and decreased anchorage dependence compared with parental CCL39 cells. Monoclonal antibodies that interfere with signal transduction by the human CSF-1 receptor inhibited both basal [3H]thymidine incorporation and soft agar colony formation, indicating that relaxation of growth control was dependent on CSF-1 receptor expression.

Both granulocyte-macrophage CSF and macrophage CSF control the proliferation and survival of the same subset of alveolar macrophages.

The effect of granulocyte-macrophage (GM)-CSF on the proliferation of murine pulmonary alveolar macrophages in vitro was investigated. About 20% of freshly isolated alveolar macrophages formed colonies in both liquid and soft agar cultures in the presence of GM-CSF. GM-CSF was also found to be capable of maintaining the survival of these colony-forming cells in vitro. Moreover, GM-CSF could substitute for CSF-1 in maintaining the survival of CSF-1-responding pulmonary alveolar macrophage colony-forming cells in the absence of CSF-1. The concentration of GM-CSF required for maintaining the survival of colony-forming cells without proliferation was much lower than that required for the proliferation of these cells in vitro. It also enhanced the CSF-1-dependent clonal growth of alveolar macrophages. These data suggest that the colony-forming cells that respond to GM-CSF are the same subset of macrophages that form colonies in the presence of CSF-1. GM-CSF did not inhibit the binding of 125I-CSF-1 to alveolar macrophages at 0 degrees C. However, the preincubation of macrophages with GM-CSF at 37 degrees C resulted in a transient down-regulation of CSF-1 binding activity.

Ligand-induced tyrosine kinase activity of the colony-stimulating factor 1 receptor in a murine macrophage cell line.

Metabolic labeling of simian virus 40-immortalized murine macrophages with 32Pi and immunoblotting with antibodies to phosphotyrosine demonstrated that the c-fms proto-oncogene product (colony-stimulating factor 1 [CSF-1] receptor) was phosphorylated on tyrosine in vivo and rapidly degraded in response to CSF-1. Stimulation of the CSF-1 receptor also induced immediate phosphorylation of several other cellular proteins on tyrosine. By contrast, the mature cell surface glycoprotein encoded by the v-fms oncogene was phosphorylated on tyrosine in the absence of CSF-1, suggesting that it functions as a ligand-independent kinase.

Ligand-induced tyrosine kinase activity of the colony-stimulating factor 1 receptor in a murine macrophage cell line.

Metabolic labeling of simian virus 40-immortalized murine macrophages with 32Pi and immunoblotting with antibodies to phosphotyrosine demonstrated that the c-fms proto-oncogene product (colony-stimulating factor 1 [CSF-1] receptor) was phosphorylated on tyrosine in vivo and rapidly degraded in response to CSF-1. Stimulation of the CSF-1 receptor also induced immediate phosphorylation of several other cellular proteins on tyrosine. By contrast, the mature cell surface glycoprotein encoded by the v-fms oncogene was phosphorylated on tyrosine in the absence of CSF-1, suggesting that it functions as a ligand-independent kinase.

Effect of bisphosphonates on proliferation and viability of mouse bone marrow-derived macrophages.

Bisphosphonates (BP) are powerful inhibitors of bone resorption. Their mechanism of action, although still unclear, is now believed to be at the cellular level. In this study we investigated the effects of these compounds on proliferation, induced either by L-cell conditioned medium (CSF-1) or 4-phorbol 12-myristate 13-acetate (PMA) of bone marrow cells (BMC) and on CSF-1-induced proliferation and viability of bone marrow derived macrophages (BMDM phi). BMC proliferation, measured by [3H]-TdR incorporation or by clonal assay in soft agar, was significantly inhibited by 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (AHBuBP) and 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (AHPrBP) at 2.5 x 10(-7) M and by dichloromethylenebisphosphonate (Cl2MBP) at 2.5 x 10(-6) M. This inhibitory effect was also confirmed on the proliferation, measured by [3H]-TdR incorporation, of BMDM phi. In the absence of CSF-1, the viability of this latter cell population, estimated by DNA content per well and lactate dehydrogenase (LDH) released into the medium, was affected in the following order of concentrations: Cl2MBP, 1.0 x 10(-4) M; AHBuBP, 5.0 x 10(-5) M; and AHPrBP, 2.5 x 10(-5) M. Since osteoclasts and macrophages might share a common early progenitor cell, probably under the control of CSF-1, the effect exerted by BP on the proliferation of the macrophage precursors may also be extended to the osteoclast precursors.