M-CSF Treatment Research Articles

M2-like macrophages transplantation protects against the doxorubicin-induced heart failure via mitochondrial transfer

AimsThe alternatively activated macrophages have shown a cardioprotective effect in heart failure. However, the effect of M2 adoptive transfer in non-ischemic heart failure is unknown. In this study, we evaluated the efficacy of M-CSF plus IL-4 induced M2-like macrophages transplantation in doxorubicin-induced cardiotoxicity.MethodsBone marrow mononuclear cells were polarized as CCR2+CD206+ M2-like macrophages by a combination of M-CSF plus IL-4 treatment. C57BL/6 mice received a single intraperitoneal injection of doxorubicin (15 mg/kg). The treatment group were treated with M2-like macrophages (1 × 10^6 cells per mouse; i.v.) once a week for 2 weeks. After 3 weeks, we examined the percentage of resident cells and cardiac function. Furthermore, we evaluated cardiac fibrosis, cardiomyocyte apoptosis and circulating inflammatory factors. Finally, we investigated the mitochondria transfer in vitro in a direct and indirect co-culture conditions.ResultsCardiac function was significantly improved in doxorubicin-induced heart failure by adoptive transfer of M2-like macrophages. Besides, M2-like macrophages treatment attenuated cardiac fibrosis and cardiomyocyte apoptosis, as well as increased the level of circulating IL-4 and Th2 response. In vitro, M2-like macrophages could transfer mitochondria to injured cardiomyocytes in a direct and indirect way.ConclusionsIn our study, adoptive transfer of M2-like macrophages could protect against the doxorubicin-induced cardiotoxicity, which may be partly attributed to mitochondria transfer. And M2-like macrophages transplantation could become a treatment for non-ischemic heart failure in the clinical practice.Graphical

Abstract 14042: Characterization of Multi-Cell-Type Engineered Heart Tissues Containing Human iPSC-Derived Cardiomyocytes, Endothelial Cells, Fibroblasts, Smooth Muscle Cells and Macrophages

Introduction: Induction of a pathological type of hypertrophy in human engineered heart tissues (EHTs) has not yet been fully achieved. We hypothesized that this limitation is caused by the unicellularity of EHTs, which contain only cardiomyocytes (CMs) and lack the interactions with non-myocytes. Thus, we aimed to generate EHTs implementing five native cardiac cell types, i.e., CMs, endothelial cells (ECs), fibroblasts (Fbs), smooth muscle cells (SMCs), and macrophages (Mϕ). Methods and Results: CMs were differentiated from hiPSCs by mesodermal induction followed by inhibition of Wnt-signaling. Mesoderm progenitors were also used to differentiate ECs via VEGF stimulation. Differentiation into SMCs and Fbs required Wnt pathway activation followed by stimulation with TGFβ+bFGF or bFGF only, respectively. Finally, Mϕ differentiation required hematopoietic/myeloid specification by IL-3 and M-CSF treatment. Successful differentiation was analyzed by electron microscopy (EM) and expression profiling of hallmark genes and proteins. For instance, Mϕs exhibited high expression of myeloid markers (98.0% CD68 + , 90.9% CD14 + ) whereas Fbs showed upregulation in genes such as POSTN, DDR2, COL1A1 (all >100 fold higher than iPSCs). Functionality of all cell types was demonstrated by multiple assays. For example, the native Mϕ function was demonstrated via degradation and phagocytosis assays while ECs exhibited fast and extensive tube formation in matrigel assays. Additionally, Fbs produced 4.6x more collagen after TGFβ stimulation than unstimulated controls. In effect, multi-cell-type EHTs were generated from 60% CMs, 20% ECs, 10% Mϕ, 5% SMCs and 5% Fbs. These EHTs were characterized by confocal microscopy, serial block-face scanning EM, gene expression profiling, and automated contractility measurements. Pilot experiments revealed that the addition of different cell types accelerated the development of EHTs which was associated with earlier onset of beating and shorter diameter of the EHTs. Conclusion: Successful application of cell differentiation protocols resulted in the integration of five cell types in a multi-cell-type EHT system. The full functional profile and reaction to a stress stimulus will be investigated in future experiments.

The influence of M-CSF on fracture healing in a mouse model

Macrophage colony-stimulating factor 1 (M-CSF) is known to play a critical role during fracture repair e.g. by recruiting stem cells to the fracture site and impacting hard callus formation by stimulating osteoclastogenesis. The aim of this experiment was to study the impact of systemic M-CSF application and its effect on bony healing in a mouse model of femoral osteotomy. Doing so, we studied 61 wild type (wt) mice (18-week-old female C57BL/6) which were divided into three groups: (1) femoral osteotomy, (2) femoral osteotomy + stabilization with external fixator and (3) femoral osteotomy + stabilization with external fixator + systemic M-CSF application. Further, 12 op/op mice underwent femoral osteotomy and served as proof of concept. After being sacrificed at 28 days bony bridging was evaluated ex vivo with µCT, histological and biomechanical testing. Systemic M-CSF application impacted osteoclasts numbers, which were almost as low as found in op/op mice. Regarding callus size, the application of M-CSF in wt mice resulted in significantly larger calluses compared to wt mice without systemic M-CSF treatment. We further observed an anabolic effect of M-CSF application resulting in increased trabecular thickness compared to wt animals without additional M-CSF application. Systemic M-CSF application did not alter biomechanical properties in WT mice. The impact of M-CSF application in a mouse model of femoral osteotomy was oppositional to what we were expecting. While M-CSF application had a distinct anabolic effect on callus size as well as trabecular thickness, this on bottom line did not improve biomechanical properties. We hypothesize that in addition to the well-recognized negative effects of M-CSF on osteoclast numbers this seems to further downstream cause a lack of feedback on osteoblasts. Ultimately, continuous M-CSF application in the absence of co-stimulatory signals (e.g. RANKL) might overstimulate the hematopoietic linage in favor of tissue macrophages instead of osteoclasts.

Characterization of Extracellular Vesicles in Osteoporotic Patients Compared to Osteopenic and Healthy Controls.

Extracellular vesicles (EVs) are mediators of a range of pathological conditions. However, their role in bone loss disease has not been well understood. In this study we characterized plasma EVs of 54 osteoporotic (OP) postmenopausal women compared to 48 osteopenic (OPN) and 44 healthy controls (CN), and we investigated their effects on osteoclasts and osteoblasts. We found no differences between the three groups in terms of anthropometric measurements and biochemical evaluation of serum calcium, phosphate, creatinine, PTH, 25-hydroxy vitamin D and bone biomarkers, except for an increase of CTX level in OP group. FACS analysis revealed that OP patients presented a significantly increased number of EVsand RANKL+ EVs compared with both CN and OPN subjects. Total EVs are negatively associated with the lumbar spine T-score and femoral neck T-score. Only in the OPN patients we observed a positive association between the total number of EVs and RANKL+ EVs with the serum RANKL. In vitro studies revealed that OP EVs supported osteoclastogenesis of healthy donor peripheral blood mononuclear cells at the same level observed following RANKL and M-CSF treatment, reduced the ability of mesenchymal stem cells to differentiate into osteoblasts, while inducing an increase of OSTERIX and RANKL expression in mature osteoblasts. The analysis of miRNome revealed that miR-1246 and miR-1224-5p were the most upregulated and downregulated in OP EVs; the modulated EV-miRNAs in OP and OPN compared to CN are related to osteoclast differentiation, interleukin-13 production and regulation of canonical WNT pathway. A proteomic comparison between OPN and CN EVs evidenced a decrease in fibrinogen, vitronectin, and clusterin and an increase in coagulation factors and apolipoprotein, which was also upregulated in OP EVs. Interestingly, an increase in RANKL+ EVs and exosomal miR-1246 was also observed in samples from patients affected by Gorham-Stout disease, suggesting that EVs could be good candidate as bone loss disease biomarkers. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

FBW7 Inhibits Myeloid Differentiation in Acute Myeloid Leukemia via GSK3-Dependent Ubiquitination of PU.1.

Glycogen synthase kinase 3β (GSK3β), an ubiquitously expressed serine/threonine kinase is reported to be overexpressed and hyperactivated in cancers including acute myeloid leukemia (AML) where it promotes self-renewal, growth, and survival of AML cells. Therefore, GSK3β inhibition results in AML cell growth inhibition and myeloid differentiation. Here we identified master transcription factor PU.1 of monocyte-macrophage differentiation pathway as potential GSK3β target. We demonstrate that GSK3β phosphorylates PU.1 at Ser41 and Ser140 leading to its recognition and subsequent ubiquitin-mediated degradation by E3 ubiquitin ligase FBW7. This GSK3-dependent degradation of PU.1 by FBW7 inhibited monocyte-macrophage differentiation. We further showed that a phospho-deficient PU.1 mutant (PU.1-S41, S140A) neither bound to FBW7 nor was degraded by it. Consequently, PU.1-S41, S140A retained its transactivation, DNA-binding ability and promoted monocyte-macrophage differentiation of U937 cells even without phorbol 12-myristate 13-acetate (PMA) treatment. We further showed that FBW7 overexpression inhibited both PMA as well as M-CSF-induced macrophage differentiation of myeloid cell lines and peripheral blood mononuclear cells (PBMC) from healthy volunteers, respectively. Contrarily, FBW7 depletion promoted differentiation of these cells even without any inducer suggesting for a robust role of GSK3β-FBW7 axis in negatively regulating myeloid differentiation. Furthermore, we also recapitulated these findings in PBMCs isolated from patients with leukemia where FBW7 overexpression markedly inhibited endogenous PU.1 protein levels. In addition, PBMCs also showed enhanced differentiation when treated with M-CSF and GSK3 inhibitor (SB216763) together compared with M-CSF treatment alone. IMPLICATIONS: Our data demonstrate a plausible mechanism behind PU.1 restoration and induction of myeloid differentiation upon GSK3β inhibition and further substantiates potential of GSK3β as a therapeutic target in AML.

Reparative macrophage transplantation for myocardial repair: a refinement of bone marrow mononuclear cell-based therapy

Reparative macrophages play an important role in cardiac repair post-myocardial infarction (MI). Bone marrow mononuclear cells (BM-MNCs) have been investigated as a donor for cell therapy but with limited clinical success. These cells, however, may be utilized as a source for reparative macrophages. This translational study aimed to establish a robust in vitro protocol to produce functional reparative macrophages from BM-MNCs and to establish pre-clinical evidence of the efficacy of reparative macrophage transplantation for the treatment of MI. Mouse BM-MNCs were treated with M-CSF plus IL-4, IL-10, TGF-β1 or combinations of these in vitro. The concomitant administration of M-CSF and IL-4 produced the highest rate and largest number of CD11b+F4/80+CD206+ reparative macrophages. Expression and secretion of tissue repair-related factors including IGF-1, TGF-β1, VEGF and IL1-ra were remarkably enhanced in reparative macrophages compared to BM-MNCs. These cells were transplanted in a mouse MI model, resulting in evident improvement in cardiac function recovery, compared to BM-MNC transplantation. Histological studies showed that reparative macrophage transplantation enhanced myocardial tissue repair including augmented microvascular formation, reduced cardiomyocyte hypertrophy and attenuated interstitial fibrosis. Moreover, survival of reparative macrophages in the heart post-transplantation was increased compared to BM-MNCs. Reparative macrophage transplantation also increased host-derived reparative macrophages in part through TGF-β secretion. In conclusion, concomitant M-CSF + IL-4 treatment effectively produced reparative macrophages from BM-MNCs in vitro. Transplantation of produced reparative macrophage achieved a superior therapeutic efficacy, compared to BM-MNC transplantation, through the enhanced quantity and quality of donor cell engraftment. Further development of this advanced cell-based therapy is warranted.

M-CSF improves protection against bacterial and fungal infections after hematopoietic stem/progenitor cell transplantation.

Myeloablative treatment preceding hematopoietic stem cell (HSC) and progenitor cell (HS/PC) transplantation results in severe myeloid cytopenia and susceptibility to infections in the lag period before hematopoietic recovery. We have previously shown that macrophage colony-stimulating factor (CSF-1; M-CSF) directly instructed myeloid commitment in HSCs. In this study, we tested whether this effect had therapeutic benefit in improving protection against pathogens after HS/PC transplantation. M-CSF treatment resulted in an increased production of mature myeloid donor cells and an increased survival of recipient mice infected with lethal doses of clinically relevant opportunistic pathogens, namely the bacteria Pseudomonas aeruginosa and the fungus Aspergillus fumigatus M-CSF treatment during engraftment or after infection efficiently protected from these pathogens as early as 3 days after transplantation and was effective as a single dose. It was more efficient than granulocyte CSF (G-CSF), a common treatment of severe neutropenia, which showed no protective effect under the tested conditions. M-CSF treatment showed no adverse effect on long-term lineage contribution or stem cell activity and, unlike G-CSF, did not impede recovery of HS/PCs, thrombocyte numbers, or glucose metabolism. These results encourage potential clinical applications of M-CSF to prevent severe infections after HS/PC transplantation.

Mice Deficient in Angiopoietin-like Protein 2 (Angptl2) Gene Show Increased Susceptibility to Bacterial Infection Due to Attenuated Macrophage Activity

Macrophages play crucial roles in combatting infectious disease by promoting inflammation and phagocytosis. Angiopoietin-like protein 2 (ANGPTL2) is a secreted factor that induces tissue inflammation by attracting and activating macrophages to produce inflammatory cytokines in chronic inflammation-associated diseases such as obesity-associated metabolic syndrome, atherosclerosis, and rheumatoid arthritis. Here, we asked whether and how ANGPTL2 activates macrophages in the innate immune response. ANGPTL2 was predominantly expressed in proinflammatory mouse bone marrow-derived differentiated macrophages (GM-BMMs) following GM-CSF treatment relative to anti-inflammatory cells (M-BMMs) established by M-CSF treatment. Expression of the proinflammatory markers IL-1β, IL-12p35, and IL-12p40 significantly decreased in GM-BMMs from Angptl2-deficient compared with wild-type (WT) mice, suggestive of attenuated proinflammatory activity. We also report that ANGPTL2 inflammatory signaling is transduced through integrin α5β1 rather than through paired immunoglobulin-like receptor B. Interestingly, Angptl2-deficient mice were more susceptible to infection with Salmonella enterica serovar Typhimurium than were WT mice. Moreover, nitric oxide (NO) production by Angptl2-deficient GM-BMMs was significantly lower than in WT GM-BMMs. Collectively, our findings suggest that macrophage-derived ANGPTL2 promotes an innate immune response in those cells by enhancing proinflammatory activity and NO production required to fight infection.

Myeloid cytokines distinctly and reversibly control LAIR-1 (CD305) expression on monocytes-mΦs/monocyte-DCs.

Abstract Cross-linking of the inhibitory immunoreceptor LAIR-1 (CD305) on the cell surface phosphorylates LAIR-1 inhibitory motifs (ITIM) and restricts the growth of monocyte-derived dendritic cells (mono-DCs). Despite the suggestion that LAIR-1’s ability to prevent unwarranted immunity is compromised in autoimmunity, much remains to be learned about the control of LAIR-1 expression. Here, we compared the ability of various myeloid cytokines to control LAIR-1 expression on the surface of monocytes/macrophages (mϕs) and mono-DCs. Treatment of freshly isolated blood monocytes from healthy donors with mono-DC growth factors (GM-CSF+IL-4/GM-CSF+IL-13) led to reduced levels of LAIR-1 at the critical monocyte-to-mono-DC juncture. In contrast, M-CSF (a steady state/M2 cytokine) sustained LAIR-1+CD14+CD33m/M+CD16+ mono-mϕs. M-CSF effects on LAIR-1 occurred under serum-supplemented and serum-free conditions. Addition of M-CSF to mono-DCs previously cultured in GM-CSF+IL-4 promptly reversed the down-regulatory effects of GM-CSF+IL-4 producing increases in LAIR-1+CD14+ cells. Conversely, addition of GM-CSF+IL-4 to M-CSF exposed cultures lowered levels of LAIR-1 and CD14. M-CSF treatment of SLE blood myeloid cells (featuring activated monocytes/mono-DCs lacking LAIR-1, CD14) produced high levels of LAIR-1 and CD14 vs GM-CSF/IL-4. Thus: 1. The expression of LAIR-1 on monocytes/mϕs and mono-DCs is distinctly and reversibly controlled by myeloid cytokines. 2. LAIR-1 expression on circulating myeloid cells in SLE may be regulated by M-CSF.

Software-assisted morphometric and phenotype analyses of human peripheral blood monocyte-derived macrophages induced by a microenvironment model of human esophageal squamous cell carcinoma.

Human macrophages play important roles in tumor promotion and are called tumor-associated macrophages (TAMs). We previously demonstrated that human esophageal squamous cell carcinomas (ESCCs) contain TAMs and that these TAMs tend to have tumor-supporting features. Here we exposed human macrophages to conditioned media of TE-series human ESCC cell lines (TECMs) to generate an ESCC extracellular stimuli-influenced TAM model. CD14(+) peripheral blood monocytes (PBMos) from healthy donors were treated with M-CSF and with additional IL-4 or TECM exposure. Morphological changes of the cells and the induction of CD163/CD204 proteins were detected in the TECM-exposed model TAMs by immunofluorescence. A software-assisted immunofluorescent cell image analysis showed increased CD163/CD204 positivity in the model TAMs and a weak to moderate positive correlation between the cytoplasmic area and the sum fluorescent intensity of CD204. Morphological changes of the cells were significantly reflected by several cytomorphometric parameters. PBMos were elongated with M-CSF treatment, then enlarged with TECM exposure. The cytoplasmic aspect ratio was decreased by M-CSF treatment and slightly increased by TECM exposure. The nuclear-cytoplasmic ratio decreased during the whole process of cell differentiation. This system is useful for quantitative assessments of TAM-like morphological changes of macrophages and the induction of CD163/CD204 in a model ESCC microenvironment.

In Vitro Generation Of Osteoclasts From Interleukin-3 (IL-3)-Dependent Mouse Hematopoietic Cells

ObjectiveThis work aims to develop a new strategy to generate murine osteoclasts in vitro using IL-3-dependent cells prepared by 6-day IL-3 treatment of murine bone marrow cells Methods1. Here, we describe an alternative method for in vitro generation of osteoclasts, which involves the use of interleukin (IL)-3-dependent murine bone marrow cells. Bone marrow cells, isolated from 6- to 8-week old C57BL/6 were cultured in α-MEM containing 10% FBS in tissue culture dishes overnight to remove stromal cells. Then, non-adherent bone marrow cells were harvested and continued in α-MEM containing 10% FBS without (control) or with IL-3 (1 ng/ml) for 6 days. While no cells survived in the control culture after the 6-day culturing, the IL-3-treated culture gave rise to a significant number of surviving cells. These IL-3-depedent cells were capable of differentiating to osteoclasts in response to M-CSF and RANKL stimulation. Moreover, these IL-3-dependent cells can be further expanded by plating them in non-treated plastic dishes followed with M-CSF treatment; they continued to survive and proliferate in non-treated plastic dishes in the presence of M-CSF for up to 4 days. After 4-day M-CSF treatment, these cells can be lifted by EDTA, and they were still able to differentiate into osteoclasts upon subsequent stimulation of M-CSF and RANKL.2. We performed the in vitro bone resorption assay, Semiquantitative Reverse Transcription (RT)-PCR, Western Analysis, Infection of Murine Bone Marrow Cells (BMCs) to test whether the osteoclasts generated from IL-3-dependent murine bone marrow cells are different from the osteoclasts generated from traditional method. Results1. IL-3 can maintain the survival of murine bone marrow cells for up to 6 days and these cells still keep their capacity to generate osteoclasts. The capacity of IL-3-dependent cells to form osteoclasts decreases with time of IL-3 treatment and IL-3 dependent cells can be further expanded by M-CSF without significant loss of the osteoclastogenic potential.2. IL-3-dependent cells can form functional osteoclasts. RANKL induces the expression of osteoclast genes in IL-3-dependent cells. RANKL activates some of RANK signaling pathways in IL-3-dependent cells. Importantly, we found that IL-3 dependent murine bone marrow cells can be infected by retrovirus encoding GFP. Conclusions1) We have developed a new strategy to generate murine osteoclasts in vitro using IL-3-dependent cells prepared by 6-day IL-3 treatment of murine bone marrow cells.2) IL-3-dependent cells can be infected by retrovirus, permitting further experimental manipulations to express or knock down genes in IL-3-dependent cells for studying the molecular mechanism controlling differentiation and proliferation of osteoclast precursors or delineating molecular events in early osteoclastogenesis. Disclosures:No relevant conflicts of interest to declare.

HIV-1 Resistant CDK2-Knockdown Macrophage-Like Cells Generated from 293T Cell-Derived Human Induced Pluripotent Stem Cells

A major challenge in studies of human diseases involving macrophages is low yield and heterogeneity of the primary cells and limited ability of these cells for transfections and genetic manipulations. To address this issue, we developed a simple and efficient three steps method for somatic 293T cells reprogramming into monocytes and macrophage-like cells. First, 293T cells were reprogrammed into induced pluripotent stem cells (iPSCs) through a transfection-mediated expression of two factors, Oct-4 and Sox2, resulting in a high yield of iPSC. Second, the obtained iPSC were differentiated into monocytes using IL-3 and M-CSF treatment. And third, monocytes were differentiated into macrophage-like cells in the presence of M-CSF. As an example, we developed HIV-1-resistant macrophage-like cells from 293T cells with knockdown of CDK2, a factor critical for HIV-1 transcription. Our study provides a proof-of-principle approach that can be used to study the role of host cell factors in HIV-1 infection of human macrophages.

HIV-1 Tat protein enhances RANKL/M-CSF-mediated osteoclast differentiation

Impaired osteoblast/osteoclast cross-talk and bone structure homeostasis resulting in osteopenia/osteoporosis are often observed in HIV seropositive patients but the causal mechanisms remain unsettled. This study analyzed the biological effects of Tat on peripheral blood monocyte-derived osteoclast differentiation. Tat enhances osteoclast differentiation and activity induced by RANKL plus M-CSF treatment increasing both the mRNA expression of specific osteoclast differentiation markers, such as cathepsin K and calcitonin receptor, and TRAP expression and activity. These Tat-related biological effects may be related, at least in part, to the induction of c-fos expression and AP-1 activity. c-fos up-regulation was triggered by Tat when cell cultures were co-treated with RANKL/M-CSF and an analysis of c-fos promoter with c-fos deletion mutant constructs disclosed specific c-fos promoter domains targeted by Tat. Together, these results show that Tat may be considered a viral factor positively modulating the osteoclastogenesis and then bone resorption activity suggesting a pathogenetic role of this viral protein in the HIV-related osteopenia/osteoporosis.

Pyrin Critical to Macrophage IL-1β Response to Francisella Challenge

Relative to monocytes, human macrophages are deficient in their ability to process and release IL-1beta. In an effort to explain this difference, we used a model of IL-1beta processing and release that is dependent upon bacterial escape into the cytosol. Fresh human blood monocytes were compared with monocyte-derived macrophages (MDM) for their IL-1beta release in response to challenge with Francisella novicida. Although both cell types produced similar levels of IL-1beta mRNA and intracellular pro-IL-1beta, only monocytes readily released processed mature IL-1beta. Baseline mRNA expression profiling of candidate genes revealed a remarkable deficiency in the pyrin gene, MEFV, expression in MDM compared with monocytes. Immunoblots confirmed a corresponding deficit in MDM pyrin protein. To determine whether pyrin levels were responsible for the monocyte/MDM difference in mature IL-1beta release, pyrin expression was knocked down by nucleofecting small interfering RNA against pyrin into monocytes or stably transducing small interfering RNA against pyrin into the monocyte cell line, THP-1. Pyrin knockdown was associated with a significant drop in IL-1beta release in both cell types. Importantly, M-CSF treatment of MDM restored pyrin levels and IL-1beta release. Similarly, the stable expression of pyrin in PMA-stimulated THP-1-derived macrophages induces caspase-1 activation, associated with increased IL-1beta release after infection with F. novicida. In summary, intracellular pyrin levels positively regulate MDM IL-1beta responsiveness to Francisella challenge.

Multiple roles of M‐CSF in human osteoclastogenesis

Although the critical role of M-CSF in osteoclastogenesis is well documented, there has been no detailed analysis of how it regulates human osteoclast formation and function in vitro. We used a human osteoclastogenesis model employing CFU-GM osteoclast precursors cultured for 14 days on dentine with RANKL, with varying exposure to exogenous human M-CSF. Short-term treatment of precursors with M-CSF (10-100 ng/mL) resulted in increased proliferation with or without RANKL. Treatment with M-CSF (1-100 ng/mL) for 14 days caused a biphasic concentration-dependent stimulation of formation, fusion, and resorption peaking at 10-50 ng/mL and almost complete abolition of resorption at 100 ng/mL. Time-course studies using M-CSF (25 ng/mL) showed that osteoclast size, nuclei/cell, and resorption increased with longer duration of M-CSF treatment. When treatment was restricted to the first 4 days, M-CSF (25-100 ng/mL) stimulated formation of normal numbers of osteoclasts that resorbed less. Blockade of endogenous M-CSF signaling with neutralizing M-CSF antibody during the first week of culture extensively inhibited osteoclastogenesis, whereas blockade during the second week produced only a small reduction in resorption. Treatment with M-CSF during the second week of culture caused a small increase in osteoclast number and a concentration-dependent increase in cytoplasmic spreading with inhibition of resorption. We have shown that M-CSF modulates multiple steps of human osteoclastogenesis, including proliferation, differentiation and fusion of precursors. In the later stages of osteoclastogenesis, M-CSF modulates osteoclast-resorbing activity, but is not required for survival. Modulation of M-CSF signaling is a potential therapeutic target for conditions associated with excess bone resorption.

Macrophage Colony-Stimulating Factor Induces Vascular Endothelial Growth Factor Production in Skeletal Muscle and Promotes Tumor Angiogenesis

Although M-CSF has been used for myelosuppression due to chemotherapy in patients with solid tumors, the effect of exogenous M-CSF on tumor angiogenesis has not been studied. In this study we showed that M-CSF has the ability to accelerate solid tumor growth by enhancing angiogenesis with a novel mechanism. M-CSF accelerated intratumoral vessel density in tumors inoculated into mice, although it did not accelerate the proliferation of malignant cells and cultured endothelial cells in vitro. In both the absence and the presence of tumors, M-CSF significantly increased the circulating cells that displayed phenotypic characteristics of endothelial progenitor cells in mice. Moreover, M-CSF treatment induced the systemic elevation of vascular endothelial growth factor (VEGF). VEGFR-2 kinase inhibitor significantly impaired the effect of M-CSF on tumor growth. In vivo, M-CSF increased VEGF mRNA expression in skeletal muscles. Even after treatment with carageenan and anti-CD11b mAb in mice, M-CSF increased VEGF production in skeletal muscles, suggesting that systemic VEGF elevation was attributed to skeletal muscle VEGF production. In vitro, M-CSF increased VEGF production and activated the Akt signaling pathway in C2C12 myotubes. These results suggest that M-CSF promotes tumor growth by increasing endothelial progenitor cells and activating angiogenesis, and the effects of M-CSF are largely based on the induction of systemic VEGF from skeletal muscles.

Focal adhesion kinase is redistributed to focal complexes and mediates cell spreading in macrophages in response to M-CSF

The macrophage colony-stimulating factor (M-CSF, CSF-1) regulates survival, proliferation and differentiation of mononuclear phagocytes, as well as macrophage motility and morphology. The latter features are usually regulated by ECM-mediated activation of integrins and subsequent tyrosine phosphorylation of cellular proteins, including focal adhesion kinase (FAK). FAK is phosphorylated by downstream receptor tyrosine kinases as well. We addressed the question whether M-CSF regulates FAK tyrosine phosphorylation in macrophages, and found that M-CSF induces FAK phosphorylation at all known tyrosine residues. This phosphorylation was dependent on Src. Extracellularly-regulated kinase (ERK), Jun N-terminal kinase (JNK) and phosphatidylinositol-3-kinase (PI3K) were found to be negatively involved in M-CSF-induced FAK phosphorylation, as their inhibition resulted in FAK hyper-phosphorylation. Following M-CSF treatment, FAK and the active forms of M-CSFR and Src were redistributed to the cytoskeleton, where active ERK, JNK and PI3K were detectable. Immunofluorescence showed the presence of FAK and its active form in focal complexes following M-CSF treatment. Moreover, cell spreading and adhesion were impaired when FAK tyrosine phosphorylation was abrogated by either transfection with FRNK, a dominant negative form of FAK, or treatment with a number of inhibitors of upstream FAK-activating signals. These results point to a relevant role for FAK in the regulation of cell spreading and adhesion in macrophages.

Vitamin D antagonist, TEI-9647, inhibits osteoclast formation induced by 1α,25-dihydroxyvitamin D 3 from pagetic bone marrow cells

(23 S)-25-Dehydro-1α-hydroxyvitamin D 3-26,23-lactone (TEI-9647) functions an antagonist of the 1α,25-dihydroxyvitamin D 3 (1α,25-(OH) 2D 3) nuclear receptor (VDR)-mediated differentiation of human leukemia (HL-60) cells [J. Biol. Chem. 274 (1999) 16392]. We examined the effect of vitamin D antagonist, TEI-9647, on osteoclast formation induced by 1α,25-(OH) 2D 3 from bone marrow cells of patients with Paget’s disease. TEI-9647 itself never induced osteoclast formation even at 10 −6 M, but dose-dependently (10 −10 to 10 −6 M) inhibited osteoclast formation induced by physiologic concentrations of 1α,25-(OH) 2D 3 (41 pg/ml, 10 −10 M) from bone marrow cells of patients with Paget’s disease. At the same time, 10 −8 M of TEI-9647 alone did not cause 1α,25-(OH) 2D 3 dependent gene expression, but almost completely suppressed TAF II-17, a potential coactivator of VDR and 25-hydroxyvitamin D 3-24-hydroxylase (25-OH-D 3-24-hydroxylase) gene expression induced by 10 −10 M 1α,25-(OH) 2D 3 in bone marrow cells of patients with Paget’s disease. Moreover, TEI-9647 dose-dependently inhibited bone resorption induced by 10 −9 M 1α,25-(OH) 2D 3 by osteoclasts produced by RANKL and M-CSF treatment of measles virus nucleocapsid gene transduced bone marrow cells. These results suggest that TEI-9647 acts directly on osteoclast precursors and osteoclasts, and that TEI-9647 may be a novel agent to suppress the excessive bone resorption and osteoclast formation in patients with Paget’s disease.

Phosphatidylinositol 3-kinase coordinately activates the MEK/ERK and AKT/NFkappaB pathways to maintain osteoclast survival.

We have examined highly purified osteoclasts that were generated in vitro from murine co-culture of marrow precursors with stromal support cells and have found evidence of activation of the MEK/ERK and AKT/NFkappaB survival pathways. Many mature marrow-derived osteoclasts survived for at least 48 h in culture whether or not they are maintained with stromal cells. Moreover, supplementing purified osteoclasts with RANKL and/or M-CSF had no impact on their survival pattern. In addition, spleen-derived osteoclasts generated with RANKL and M-CSF treatment exhibited a similar survival pattern. Blocking MEK, AKT, or NFkappaB activity resulted in apoptosis of many, but not all, of the osteoclasts in purified marrow-derived osteoclasts, marrow-derived osteoclasts co-cultured with stromal cells, and spleen-derived osteoclasts maintained with RANKL and M-CSF. These data support that both the MEK/ERK and AKT/NFkappaB pathways contribute to osteoclast survival. Since PI3K has been shown to activate either of these pathways, we have examined its role in osteoclast survival. PI3K inhibition caused apoptosis of nearly all osteoclasts in purified and co-cultured marrow-derived osteoclasts and spleen-derived osteoclasts maintained with RANKL and M-CSF. Interestingly, in marrow-derived co-cultures, the apoptotic response was restricted to osteoclasts as there was no evidence of stromal support cell apoptosis. PI3K inhibition also blocked MEK1/2, ERK1/2, and AKT phosphorylation and NFkappaB activation in purified osteoclasts. Simultaneous blockage of both AKT and MEK1/2 caused rapid apoptosis of nearly all osteoclasts, mimicking the response to PI3K inhibition. These data reveal that PI3K coordinately activates two distinct survival pathways that are both important in osteoclast survival.

Macrophage colony-stimulating factor restored chemotherapy-induced granulocyte dysfunctions: role of IL-8 production by monocytes

We evaluated the influence of M-CSF treatment on granulocyte functions in patients with ovarian cancer. Eighteen patients with ovarian cancer received two consecutive courses of chemotherapy (16 cases, CAP therapy and two cases, CP therapy) at 4-week intervals. M-CSF (8 million U/day) was infused for 7 days starting from the next day after chemotherapy. Superoxide anion production by isolated peripheral blood granulocytes, their phagocytosis, and expression of cell adhesion molecules such as CD11a, CD11b, and CD18 on granulocytes were measured by flow cytometry. Cytokine (IL-8, G-CSF, and GM-CSF) levels in peripheral blood monocyte (PBM) culture supernatants were measured by enzyme-linked immunosorbent assay in 5 out of 18 cases. The levels of CD11a, CD11b and CD18 expression on peripheral blood granulocytes and superoxide anion production by granulocytes were significantly suppressed by chemotherapy without CSF support. The levels of CD11a and CD18 expression on granulocytes were significantly enhanced by administration of M-CSF. When M-CSF was added to cultured PBM, the level of IL-8 in the supernatant increased with the concentration of M-CSF. When IL-8 was added to cultured granulocytes, the levels of CD18 expression on granulocytes and superoxide anion production by granulocytes were significantly increased. These observations suggest that M-CSF enhances the production of IL-8 from monocytes in vivo, thereby improving chemotherapy-induced granulocyte dysfunction.