Colony-stimulating Factor-1 Receptor Signaling Research Articles

Control of macrophage lineage populations by CSF‐1 receptor and GM‐CSF in homeostasis and inflammation

There is recent interest in the role of monocyte/macrophage subpopulations in pathology. How the hemopoietic growth factors, macrophage-colony stimulating factor (M-CSF or CSF-1) and granulocyte macrophage (GM)-CSF, regulate their in vivo development and function is unclear. A comparison is made here on the effect of CSF-1 receptor (CSF-1R) and GM-CSF blockade/depletion on such subpopulations, both in the steady state and during inflammation. In the steady state, administration of neutralizing anti-CSF-1R monoclonal antibody (mAb) rapidly (within 3-4 days) lowered, specifically, the number of the more mature Ly6C(lo) peripheral blood murine monocyte population and resident peritoneal macrophages; it also reduced the accumulation of murine exudate (Ly6C(lo)) macrophages in two peritonitis models and alveolar macrophages in lung inflammation, consistent with a non-redundant role for CSF-1 (or interleukin-34) in certain inflammatory reactions. A neutralizing mAb to GM-CSF also reduced inflammatory macrophage numbers during antigen-induced peritonitis and lung inflammation. In GM-CSF gene-deficient mice, a detailed kinetic analysis of monocyte/macrophage and neutrophil dynamics in antigen-induced peritonitis suggested that GM-CSF was acting, in part, systemically to maintain the inflammatory reaction. A model is proposed in which CSF-1R signaling controls the development of the macrophage lineage at a relatively late stage under steady state conditions and during certain inflammatory reactions, whereas in inflammation, GM-CSF can be required to maintain the response by contributing to the prolonged extravasation of immature monocytes and neutrophils. A correlation has been observed between macrophage numbers and the severity of certain inflammatory conditions, and it could be that CSF-1 and GM-CSF contribute to the control of these numbers in the ways proposed.

Phosphorylation of CSF-1R Y721 mediates its association with PI3K to regulate macrophage motility and enhancement of tumor cell invasion

Colony stimulating factor-1 (CSF-1) regulates macrophage morphology and motility, as well as mononuclear phagocytic cell proliferation and differentiation. The CSF-1 receptor (CSF-1R) transduces these pleiotropic signals through autophosphorylation of eight intracellular tyrosine residues. We have used a novel bone-marrow-derived macrophage cell line system to examine specific signaling pathways activated by tyrosine-phosphorylated CSF-1R in macrophages. Screening of macrophages expressing a single species of CSF-1R with individual tyrosine-to-phenylalanine residue mutations revealed striking morphological alterations upon mutation of Y721. M⁻/⁻.Y721F cells were apolar and ruffled poorly in response to CSF-1. Y721-P-mediated CSF-1R signaling regulated adhesion and actin polymerization to control macrophage spreading and motility. Moreover, the reduced motility of M⁻/⁻.Y721F macrophages was associated with their reduced capacity to enhance carcinoma cell invasion. Y721 phosphorylation mediated the direct association of the p85 subunit of phosphoinositide 3-kinase (PI3K) with the CSF-1R, but not that of phospholipase C (PLC) γ2, and induced polarized PtdIns(3,4,5)P₃ production at the putative leading edge, implicating PI3K as a major regulator of CSF-1-induced macrophage motility. The Y721-P-motif-based motility signaling was at least partially independent of both Akt and increased Rac and Cdc42 activation but mediated the rapid and transient association of an unidentified ~170 kDa phosphorylated protein with either Rac-GTP or Cdc42-GTP. These studies identify CSF-1R-Y721-P-PI3K signaling as a major pathway in CSF-1-regulated macrophage motility and provide a starting point for the discovery of the immediate downstream signaling events.

Targeting distinct tumor-infiltrating myeloid cells by inhibiting CSF-1 receptor: combating tumor evasion of antiangiogenic therapy

AbstractTumor-infiltrating myeloid cells (TIMs) support tumor growth by promoting angiogenesis and suppressing antitumor immune responses. CSF-1 receptor (CSF1R) signaling is important for the recruitment of CD11b+F4/80+ tumor-associated macrophages (TAMs) and contributes to myeloid cell-mediated angiogenesis. However, the impact of the CSF1R signaling pathway on other TIM subsets, including CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs), is unknown. Tumor-infiltrating MDSCs have also been shown to contribute to tumor angiogenesis and have recently been implicated in tumor resistance to antiangiogenic therapy, yet their precise involvement in these processes is not well understood. Here, we use the selective pharmacologic inhibitor of CSF1R signaling, GW2580, to demonstrate that CSF-1 regulates the tumor recruitment of CD11b+Gr-1loLy6Chi mononuclear MDSCs. Targeting these TIM subsets inhibits tumor angiogenesis associated with reduced expression of proangiogenic and immunosuppressive genes. Combination therapy using GW2580 with an anti–VEGFR-2 antibody synergistically suppresses tumor growth and severely impairs tumor angiogenesis along with reverting at least one TIM-mediated antiangiogenic compensatory mechanism involving MMP-9. These data highlight the importance of CSF1R signaling in the recruitment and function of distinct TIM subsets, including MDSCs, and validate the benefits of targeting CSF1R signaling in combination with antiangiogenic drugs for the treatment of solid cancers.

High-Density Gene Expression Analysis of Tumor-Associated Macrophages from Mouse Mammary Tumors

Clinical and experimental evidence indicates that tumor-associated macrophages (TAMs) promote malignant progression. In breast cancer, TAMs enhance tumor angiogenesis, tumor cell invasion, matrix remodeling, and immune suppression against the tumor. In this study, we examined late-stage mammary tumors from a transgenic mouse model of breast cancer. We used flow cytometry under conditions that minimized gene expression changes to isolate a rigorously defined TAM population previously shown to be associated with invasive carcinoma cells. The gene expression signature of this population was compared with a similar population derived from spleens of non-tumor-bearing mice using high-density oligonucleotide arrays. Using stringent selection criteria, transcript abundance of 460 genes was shown to be differentially regulated between the two populations. Bioinformatic analyses of known functions of these genes indicated that formerly ascribed TAM functions, including suppression of immune activation and matrix remodeling, as well as multiple mediators of tumor angiogenesis, were elevated in TAMs. Further bioinformatic analyses confirmed that a pure and valid TAM gene expression signature in mouse tumors could be used to assess expression of TAMs in human breast cancer. The data derived from these more physiologically relevant autochthonous tumors compared with previous studies in tumor xenografts suggest tactics by which TAMs may regulate tumor angiogenesis and thus provide a basis for exploring other transcriptional mediators of TAM trophic functions within the tumor microenvironment.

Distinct roles of class IA PI3K isoforms in primary and immortalised macrophages

The class IA isoforms of phosphoinositide 3-kinase (p110alpha, p110beta and p110delta) often have non-redundant functions in a given cell type. However, for reasons that are unclear, the role of a specific PI3K isoform can vary between cell types. Here, we compare the relative contributions of PI3K isoforms in primary and immortalised macrophages. In primary macrophages stimulated with the tyrosine kinase ligand colony-stimulating factor 1 (CSF1), all class IA PI3K isoforms participate in the regulation of Rac1, whereas p110delta selectively controls the activities of Akt, RhoA and PTEN, in addition to controlling proliferation and chemotaxis. The prominent role of p110delta in these cells correlates with it being the main PI3K isoform that is recruited to the activated CSF1 receptor (CSF1R). In immortalised BAC1.2F5 macrophages, however, the CSF1R also engages p110alpha, which takes up a more prominent role in CSF1R signalling, in processes including Akt phosphorylation and regulation of DNA synthesis. Cell migration, however, remains dependent mainly on p110delta. In other immortalised macrophage cell lines, such as IC-21 and J774.2, p110alpha also becomes more prominently involved in CSF1-induced Akt phosphorylation, at the expense of p110delta.These data show that PI3K isoforms can be differentially regulated in distinct cellular contexts, with the dominant role of the p110delta isoform in Akt phosphorylation and proliferation being lost upon cell immortalisation. These findings suggest that p110delta-selective PI3K inhibitors may be more effective in inflammation than in cancer.

Modulation of dendritic cell differentiation by colony-stimulating factor-1: role of phosphatidylinositol 3′-kinase and delayed caspase activation

Monocytes acquire a dendritic cell (DC) phenotype when cultured with GM-CSF and IL-4. By contrast, CSF-1 is a potent inducer of monocyte-to-macrophage differentiation. Increasing evidence indicates that DC development is impaired in conditions characterized by CSF-1 overproduction, including pregnancy, trauma, and diverse malignancies. To study this, we have exposed newly established monocyte-derived DC cultures to conditions of CSF-1 excess. As a consequence, differentiation is skewed toward a unique intermediate phenotype, which we have termed DC-M. Such cells exhibit macrophage-like morphology with impaired allostimulatory capacity, altered cytokine production, and a distinctive cell surface immunophenotype. In light of the emerging role of caspase activation during macrophage differentiation, the activity of caspases 3, 8, and 9 was examined in DC and DC-M cultures. It is striking that DC-M cultures exhibit a delayed and progressive increase in activation of all three caspases, associated with depolarization of mitochondrial membrane potential. Furthermore, when DC-M cultures were supplemented with an inhibitor of caspase 8 or caspase 9, impairment of DC differentiation by CSF-1 was counteracted. To investigate upstream regulators of caspase activation in DC-M cultures, experiments were performed using inhibitors of proximal CSF-1 receptor signaling. These studies demonstrated that the PI-3K inhibitors, wortmannin and LY294002, antagonize the ability of CSF-1 to inhibit DC differentiation and to promote caspase activation. Together, these data identify a novel, PI-3K-dependent pathway by which CSF-1 directs delayed caspase activation in monocytes and thereby modulates DC differentiation.

The Src-like Adaptor Protein 2 Regulates Colony-stimulating Factor-1 Receptor Signaling and Down-regulation

Src-like adaptor protein 2 (SLAP-2) is a hematopoietic adaptor protein previously implicated as a negative regulator of T-cell antigen receptor (TCR)-mediated signaling. SLAP-2 contains an SH3 and an SH2 domain, followed by a unique carboxyl-terminal tail, which is important for c-Cbl binding. Here we describe a novel role for SLAP-2 in regulation of the colony-stimulating factor 1 receptor (CSF-1R), a receptor tyrosine kinase important for growth and differentiation of myeloid cells. SLAP-2 co-immunoprecipitates with c-Cbl and CSF-1R in primary bone marrow-derived macrophages. Using murine myeloid cells expressing CSF-1R (FD-Fms cells), we show that SLAP-2 is tyrosine-phosphorylated upon stimulation with CSF-1 and associates constitutively with both c-Cbl and CSF-1R. In addition, we show that expression of a dominant negative form of SLAP-2 impairs c-Cbl association with the CSF-1R and receptor ubiquitination. Impaired c-Cbl recruitment also correlated with changes in the kinetics of CSF-1R down-regulation and trafficking. CSF-1-mediated differentiation of FD-Fms cells and activation of downstream signaling events was also enhanced in cells stably expressing dominant negative SLAP-2. Together, these results demonstrate that SLAP-2 plays a role in c-Cbl-dependent down-regulation of CSF-1R signaling.

Inhibition of phosphorylation of the colony-stimulating factor-1 receptor (c-Fms) tyrosine kinase in transfected cells by ABT-869 and other tyrosine kinase inhibitors

The properties of several multitargeted receptor tyrosine kinase inhibitors have been studied for their inhibition of colony-stimulating factor-1 receptor (CSF-1R) signaling. A structurally novel, multitargeted tyrosine kinase inhibitor (ABT-869), imatinib (STI571), and four compounds currently in clinical development (AG013736, BAY 43-9006, CHIR258, and SU11248) were tested for inhibition of CSF-1R signaling in both the enzymatic and cellular assays. ABT-869 showed potent CSF-1R inhibition in both the enzyme and cell-based assays (IC50s < 20 nmol/L). In contrast to a previous report, we have found that imatinib has activity against human CSF-1R in both assays at submicromolar concentrations. In enzyme assays, we have found that the inhibition of CSF-1R by both ABT-869 and imatinib are competitive with ATP, with Ki values of 3 and 120 nmol/L, respectively. SU11248 is a potent inhibitor of CSF-1R in the enzyme assay (IC50 = 7 nmol/L) and inhibits receptor phosphorylation in the cellular assay (IC50 = 61 nmol/L). AG013736 was also a potent inhibitor of CSF-1R in both assays (enzyme, IC50 = 16 nmol/L; cellular, IC50 = 21 nmol/L), whereas BAY 43-9006 is less potent in the enzyme assay (IC50 = 107 nmol/L) than in the cellular system (IC50 = 20 nmol/L). In contrast, we found that CHIR258 had less activity in the cellular assay (IC50 = 535 nmol/L) relative to its enzymatic potency (IC50 = 26 nmol/L). These results show the use of a cell-based assay to confirm the inhibitory activity of lead compounds and drug candidates, such as ABT-869, against the CSF-1R protein in situ.

LPS regulates a set of genes in primary murine macrophages by antagonising CSF-1 action

We previously reported that bacterial products such as LPS and CpG DNA down-modulated cell surface levels of the Colony Stimulating Factor (CSF)-1 receptor (CSF-1R) on primary murine macrophages in an all-or-nothing manner. Here we show that the ability of bacterial products to down-modulate the CSF-1R rendered bone marrow-derived macrophages (BMM) unresponsive to CSF-1 as assessed by Akt and ERK1/2 phosphorylation. Using toll-like receptor ( tlr) 9 as a model CSF-1-repressed gene, we show that LPS induced tlr9 expression in BMM only when CSF-1 was present, suggesting that LPS relieves CSF-1-mediated inhibition to induce gene expression. Using cDNA micro-arrays, we identified a cluster of similarly CSF-1 repressed genes in BMM. By real time PCR we confirmed that the expression of a selection of these genes, including integral membrane protein 2B ( itm2b), receptor activity-modifying protein 2 ( ramp2) and macrophage-specific gene 1 ( mpg-1), were repressed by CSF-1 and were induced by LPS only in the presence of CSF-1. This pattern of gene regulation was also apparent in thioglycollate-elicited peritoneal macrophages (TEPM). LPS also counteracted CSF-1 action to induce mRNA expression of a number of transcription factors including interferon consensus sequence binding protein 1 (Icsbp1), suggesting that this mechanism leads to transcriptional reprogramming in macrophages. Since the majority of in vitro studies on macrophage biology do not include CSF-1, these genes represent a set of previously uncharacterised LPS-inducible genes. This study identifies a new mechanism of macrophage activation, in which LPS (and other toll-like receptor agonists) regulate gene expression by switching off the CSF-1R signal. This finding also provides a biological relevance to the well-documented ability of macrophage activators to down-modulate surface expression of the CSF-1R.

Macrophages Promote the Invasion of Breast Carcinoma Cells via a Colony-Stimulating Factor-1/Epidermal Growth Factor Paracrine Loop

Previous studies have shown that macrophages and tumor cells are comigratory in mammary tumors and that these cell types are mutually dependent for invasion. Here we show that macrophages and tumor cells are necessary and sufficient for comigration and invasion into collagen I and that this process involves a paracrine loop. Macrophages express epidermal growth factor (EGF), which promotes the formation of elongated protrusions and cell invasion by carcinoma cells. Colony stimulating factor 1 (CSF-1) produced by carcinoma cells promotes the expression of EGF by macrophages. In addition, EGF promotes the expression of CSF-1 by carcinoma cells thereby generating a positive feedback loop. Disruption of this loop by blockade of either EGF receptor or CSF-1 receptor signaling is sufficient to inhibit both macrophage and tumor cell migration and invasion.

Development of a quantitative, high-throughput cell-based enzyme-linked immunosorbent assay for detection of colony-stimulating factor-1 receptor tyrosine kinase inhibitors

Inhibitors of receptor tyrosine kinases are implicated as therapeutic agents for the treatment of many human diseases including cancer, inflammation and diabetes. Cell-based assays to examine inhibition of receptor tyrosine kinase mediated intracellular signaling are often laborious and not amenable to high-throughput cell-based screening of compound libraries. Here we describe the development of a nonradioactive, sandwich enzyme-linked immunosorbent assay (ELISA) to quantify the activation and inhibition of ligand-induced phosphorylation of the colony-stimulating factor-1 receptor (CSF-1R) in 96-well microtiter plate format. The assay involves the capture of the Triton X-100 solubilized human CSF-1R, from HEK293E cells overexpressing histidine epitope-tagged CSF-1R (CSF-1R/HEK293E), with immobilized CSF-1R antibody and detection of phosphosphorylation of the activated receptor with a phosphotyrosine specific antibody. The assay exhibited a 5-fold increase in phosphorylated CSF-1R signal from CSF-1R/HEK293E cells treated with colony-stimulating factor (CSF-1) relative to treated vector control cells. Additionally, using a histidine epitope-specific capture antibody, this method can also be adapted to quantify the phosphorylation state of any recombinantly expressed, histidine-tagged receptor tyrosine kinase. This method is a substantial improvement in throughput and quantitation of CSF-1R phosphorylation over conventional immunoblotting techniques.

A novel 110 kDa form of myosin XVIIIA (MysPDZ) is tyrosine-phosphorylated after colony-stimulating factor-1 receptor signalling

Macrophage colony-stimulating factor (M-CSF or CSF-1) controls the development of macrophage lineage cells via activation of its tyrosine kinase receptor, c-Fms. After adding CSF-1 to M1 myeloid cells expressing CSF-1R (CSF-1 receptor), tyrosine phosphorylation of many cellular proteins occurs, which might be linked to subsequent macrophage differentiation. The biological significance and characterization of such proteins were explored by a dual strategy comprising two-dimensional SDS/PAGE analysis of cell lysates of CSF-1-treated M1 cells expressing the wild-type or a mutated receptor, together with an enrichment strategy involving a tyrosine-phosphorylated receptor construct. In the present study, we report the identification by MS of a novel, low-abundance, 110 kDa form of myosin XVIIIA (MysPDZ, myosin containing PDZ domain), which appears to be preferentially tyrosine-phosphorylated after CSF-1R activation when compared with other known isoforms. Receptor mutation studies indicate that CSF-1R-dependent tyrosine phosphorylation of p110myosin XVIIIA requires Tyr-559 in the cytoplasmic domain of the receptor and is therefore Src-family kinase-dependent. Gelsolin, Erp61 protein disulphide-isomerase and possibly non-muscle myosin IIA were also tyrosine-phosphorylated under similar conditions. Similar to the more abundant p190 isoform, p110 myosin XVIIIA lacks a PDZ domain and, in addition, it may lack motor activity. The phosphorylation of p110 myosin XVIIIA by CSF-1 may alter its cellular localization or target its association with other proteins.

YW1-01 Colony Stimulating factor-1 receptor signaling enhances tumorigenesis and invasiveness of prostrate cancer cells (Young Urologist Research Workshop)

YW1-01 Colony Stimulating factor-1 receptor signaling enhances tumorigenesis and invasiveness of prostrate cancer cells (Young Urologist Research Workshop)

Accumulation of c-Cbl and rapid termination of colony-stimulating factor 1 receptor signaling in interferon consensus sequence binding protein-deficient bone marrow-derived macrophages.

Mice deficient for the transcription factor interferon consensus sequence binding protein (ICSBP) are immunodeficient and develop granulocytic leukemia. Further analyses indicated that ICSBP is a molecular switch factor directing the differentiation of bipotential myeloid precursors to the monocytic lineage. To reveal the molecular mechanisms responsible for the deregulation of myelopoiesis, we examined the signaling of the colony-stimulating factor 1 receptor (CSF-1R) in bone marrow-derived macrophages (BMMs) from ICSBP(-/-) mice. We found that in the absence of ICSBP, CSF-1R signaling is attenuated as seen from an accelerated termination of Erk phosphorylation and reduced cell growth. This finding coincides with an increased CSF-1R ubiquitination and an enhanced accumulation of c-Cbl. c-Cbl is an ubiquitin-ligase known to down-regulate activated CSF-1R by targeting it to the endocytic pathway. Our results indicate that upon CSF-1R activation, c-Cbl itself is partly proteolytically degraded in ICSBP(+/+) but not in ICSBP(-/-) BMMs. Congruently, the expression of a major endosomal/lysosomal protease, cathepsin B, is strongly reduced in ICSBP(-/-) BMMs.

Expression of colony stimulating factor-1 receptor (CSF-1R) by CNS neurons in mice

We report that neurons in the central nervous system express colony stimulating factor-1 receptor (CSF-1R) mRNA and protein and that the expression has regional specificity. The presence of CSF-1R in neurons was demonstrated by the use of four different types of antibodies to CSF-1R and the presence of CSF-1R mRNA by in situ hybridization using oligonucleotide probe. In the steady state in most areas of the brain, CSF-1R is weakly expressed in only a few neurons. In the cerebellum, brainstem, and spinal cord, however, CSF-1R is expressed constitutively in greater numbers of neurons. After cerebral cortex ischemic injury, neurons in the area next to the ischemic lesion markedly upregulate CSF-1R. It is also upregulated in the contralateral cortex and in many other areas of the brain and spinal cord. We demonstrated that in cultures the ligand CSF-1 binds to its receptor (CSF-1R) in neurons and that reduction of the number of apoptotic neurons and potentiation of neuron survival is CSF-1 dose dependent. We propose that CSF-1/CSF-1R signaling is an important regulatory pathway between neurons, microglia, and astrocytes.

Expression of human colony-stimulating factor-1 (CSF-1) receptor in murine pluripotent hematopoietic NFS-60 cells induces long-term proliferation in response to CSF-1 without loss of erythroid differentiation potential

NFS-60 and FDCP-Mix cells are interleukin-3--dependent multipotent hematopoietic cells that can differentiate in vitro into mature myeloid and erythroid cells. Retrovirus-mediated transfer of the human colony- stimulating factor-1 (CSF-1) receptor gene (c-fms) enabled NFS-60 cells but not FDCP-Mix cells to proliferate in response to CSF-1. The phenotype of NFS-60 cells expressing the human CSF-1 receptor (CSF-1R) grown in CSF-1 did not grossly differ from that of original NFS-60 as assessed by cytochemical and surface markers. Importantly, these cells retained their erythroid potentiality. In contrast, a CSF-1-dependent variant of NFS-60, strongly expressing murine CSF-1R, differentiated into monocyte/macrophages upon CSF-1 stimulation and almost totally lost its erythroid potentiality. We also observed that NFS-60 but not FDCP-Mix cells could grow in response to stem cell factor, (SCF), although both cell lines express relatively high amounts of SCF receptors. This suggests that SCF-R and CSF-1R signalling pathways share at least one component that may be missing or insufficiently expressed in FDCP-Mix cells. Taken together, these results suggest that human CSF-1R can use the SCF-R signalling pathway in murine multipotent cells and thereby favor self-renewal versus differentiation.