Granulocyte-macrophage Colony-stimulating Factor Stimulation Research Articles

Mesenchymal Stem/Stromal Cells Induce Myeloid-Derived Suppressor Cells in the Bone Marrow via the Activation of the c-Jun N-Terminal Kinase Signaling Pathway.

Our previous study demonstrated that mesenchymal stem/stromal cells (MSCs) induce the differentiation of myeloid-derived suppressor cells (MDSCs) in the bone marrow (BM) under inflammatory conditions. In this study, we aimed to investigate the signaling pathway involved. RNA-seq revealed that the mitogen-activated protein kinase (MAPK) pathway exhibited the highest number of upregulated genes in MSC-induced MDSCs. Western blot analysis confirmed the strong phosphorylation of c-Jun N-terminal kinase (JNK) in BM cells cocultured with MSCs under granulocyte-macrophage colony-stimulating factor stimulation, whereas p38 kinase activation remained unchanged in MSC-cocultured BM cells. JNK inhibition by SP600125 abolished the expression of Arg1 and Nos2, hallmark genes of MDSCs, as well as Hif1a, a molecule mediating monocyte functional reprogramming toward a suppressive phenotype, in MSC-cocultured BM cells. JNK inhibition also abrogated the effects of MSCs on the production of TGF-β1, TGF-β2 and IL-10 in BM cells. Furthermore, JNK inhibition increased Tnfa expression, while suppressing IL-10 production, in MSC-cocultured BM cells in response to lipopolysaccharides. Collectively, our results suggest that MSCs induce MDSC differentiation and promote immunoregulatory cytokine production in BM cells during inflammation, at least in part, through the activation of the JNK-MAPK signaling pathway.

Investigating Gene Expression of Bone-Marrow Derived Macrophages with GM-CSF Stimulation in vitro

Macrophages are specialized immune cells that play a crucial role in engulfing and destroying harmful pathogens and debris within the body. When stimulated with specific cytokines, macrophages transition into subtypes with specialized functions. For example, macrophages can differentiate into classically activated macrophages (M1) when stimulated with interferon-γ (IFN-γ ) and lipopolysaccharide (LPS). M1 macrophages are a subtype of activated macrophages that exhibit pro-inflammatory properties and are essential for combating infections and promoting tissue inflammation and repair. When macrophages are stimulated with another cytokine, known as Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), they tend to develop an M1-like phenotype. The altered gene expression that occurs when macrophages are cultured in GM-CSF remains a relatively unexplored area, with few studies having profiled this modified genotype of bone marrow-derived macrophages (BMDMs). This study aims to characterize the genotype of GM-CSF macrophages using an in vitro model as well as a genetic database. Expression data from M-CSF or GM-CSF BMDM was retrieved using microarrays, and differential gene analysis was performed using R studio. The analysis suggested an increase in pro-inflammatory gene expression in GM-CSF BMDMs compared to M-CSF-induced macrophages. The findings are reinforced by RT-PCR and qPCR analyses of specific key pro-inflammatory genes. For instance, interleukin-6 (IL-6), IL-1, as well as NLR family pyrin containing domain 3 (NLRP3) were all significantly upregulated in GM-CSF BMDMs. This project seeks to comprehensively characterize GM-CSF-induced macrophages as pro-inflammatory agents. Moreover, the cytokine's propensity to induce a pro-inflammatory response raises concerns about its applicability in treatment plans.

Granulocyte-macrophage colony-stimulating factor-stimulated human macrophages demonstrate enhanced functions contributing to T-cell activation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been implicated in numerous chronic inflammatory diseases, including multiple sclerosis (MS). GM-CSF impacts multiple properties and functions of myeloid cells via species-specific mechanisms. Therefore, we assessed the effect of GM-CSF on different human myeloid cell populations found in MS lesions: monocyte-derived macrophages (MDMs) and microglia. We previously reported a greater number of interleukin (IL)-15+ myeloid cells in the brain of patients with MS than in controls. Therefore, we investigated whether GM-CSF exerts its deleterious effects in MS by increasing IL-15 expression on myeloid cells. We found that GM-CSF increased the proportion of IL-15+ cells and/or IL-15 levels on nonpolarized, M1-polarized and M2-polarized MDMs from healthy donors and patients with MS. GM-CSF also increased IL-15 levels on human adult microglia. When cocultured with GM-CSF-stimulated MDMs, activated autologous CD8+ T lymphocytes secreted and expressed significantly higher levels of effector molecules (e.g. interferon-γ and GM-CSF) compared with cocultures with unstimulated MDMs. However, neutralizing IL-15 did not attenuate enhanced effector molecule expression on CD8+ T lymphocytes triggered by GM-CSF-stimulated MDMs. We showed that GM-CSF stimulation of MDMs increased their expression of CD80 and ICAM-1 and their secretion of IL-6, IL-27 and tumor necrosis factor. These molecules could participate in boosting the effector properties of CD8+ T lymphocytes independently of IL-15. By contrast, GM-CSF did not alter CD80, IL-27, tumor necrosis factor and chemokine (C-X-C motif) ligand 10 expression/secretion by human microglia. Therefore, our results underline the distinct impact of GM-CSF on human myeloid cells abundantly present in MS lesions.

Unintended Observations Leading to Macrophage Growth and Neutrophil Factor Research

My research area in the pharmaceutical industry is innate immunity, especially in phagocytic cells. First, I studied the heat-stable growth factor of peripheral macrophages in tumorous ascitic fluid and found that lipoproteins are an influencing factor. Later, my colleagues and I found that lipid-containing substances, namely, oxidized low-density lipoprotein, dead neutrophils, or purified lipids that could be scavenged by macrophages, induce their growth. From the series of this study, I concluded that phagocytic substances induce macrophage growth by autocrine stimulation of granulocyte-macrophage colony-stimulating factor (GM-CSF). During the study, we found that neutrophils have growth-inhibitory effects against a variety of cells. Then, I elucidated that the primary factor is a zinc-binding protein, calprotectin, an abundant protein complex in the neutrophil cytosol. I found that calprotectin induces apoptosis in many cell types, including tumor cells and normal fibroblasts, and that the zinc-binding capacity is essential for its activity. Microscopic observations revealed that neutrophil extract contains factor-inducing three-dimensional cell aggregation of human mammary carcinoma, MCF-7. I elucidated that cathepsin G is responsible for this activity and that its effect is dependent on the activation of insulin-like growth factor-1. I believe that this modest, albeit novel, observation was crucial to my thirty-nine-year-long career researching phagocytic cells.

Dexamethasone Attenuates Oncostatin M Production via Suppressing of PI3K/Akt/NF-κB Signaling in Neutrophil-like Differentiated HL-60 Cells.

Oncostatin M (OSM) plays a role in various inflammatory reactions, and neutrophils are the main source of OSM in pulmonary diseases. However, there is no evidence showing the mechanism of OSM production in neutrophils. While dexamethasone (Dex) has been known to exert anti-inflammatory activity in various fields, the precise mechanisms of OSM downregulation by Dex in neutrophils remain to be determined. Here, we examined how OSM is produced in neutrophil-like differentiated HL-60 cells. Enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and Western blot analysis were utilized to assess the potential of Dex. Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation resulted in OSM elevation in neutrophil-like dHL-60 cells. OSM elevation induced by GM-CSF is regulated by phosphatidylinositol 3-kinase (PI3K)/Akt/nuclear factor (NF)-kB signal cascades. GM-CSF stimulation upregulated phosphorylated levels of PI3K or Akt or NF-κB in neutrophil-like dHL-60 cells. Treatment with Dex decreased OSM levels as well as the phosphorylated levels of PI3K or Akt or NF-κB in neutrophil-like dHL-60 cells. Our findings show the potential of Dex in the treatment of inflammatory diseases via blocking of OSM.

G-CSF as a suitable alternative to GM-CSF to boost dinutuximab-mediated neutrophil cytotoxicity in neuroblastoma treatment

BackgroundCurrent immunotherapy for patients with high-risk neuroblastoma involves the therapeutic antibody dinutuximab that targets GD2, a ganglioside expressed on the majority of neuroblastoma tumors. Opsonized tumor cells are killed through...

Elevated chemokines and cytokines for eosinophils in neuromyelitis optica spectrum disorders

Background: Eosinophil infiltration is one of the distinctive features in neuromyelitis optica spectrum disorders (NMOSD) but not in other demyelinating diseases including multiple sclerosis (MS). Eosinophils express the chemokine receptor CCR3, which is activated by eotaxins (eotaxin-1, -2, and -3) and monocyte chemoattractant protein (MCP)-4. We aimed to investigate the role of MCPs (MCP-1, -2, -3, and -4) and eotaxins in the acute phase of NMOSD. Methods: Levels of serum and cerebrospinal fluid (CSF) eotaxins, MCPs, interleukin (IL)-5, tumor necrosis factor (TNF)-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-6 were measured using the cytokine multiplex assay from 26 patients with NMOSD (13 with immunotherapy, 13 without immunotherapy), 9 patients with MS, and 9 patients with other noninflammatory neurological diseases (OND). Glial fibrillary acidic protein was assessed using ELISA. Results: Serum MCP-1 and CSF MCP-2 levels were significantly higher in patients with NMOSD than in OND. Moreover, serum MCP-4 and CSF eotaxin-2 and -3 levels were significantly higher in NMOSD patients compared to MS and OND. Serum MCP-1, -4 and CSF eotaxin-2, -3 levels were significantly correlated with the Expanded Disability Status Scale in NMOSD. TNF-α and GM-CSF, which stimulate the above chemokines, were higher in patients with NMOSD than those in OND. Moreover, serum MCP-1 and -4 were significantly increased by IL-5 and GM-CSF stimulation, but not by TNF-α and IL-6. Only CSF eotaxin-2 was significantly increased by GM-CSF. There were no significant differences in serum MCP-1 and -4 levels between NMOSD patients with and without immunotherapy. Conclusion: These findings suggest that the elevated serum MCP-1, -4 and CSF eotaxin-2, -3 may be a key step in eosinophil recruitment in the acute phase of NMOSD.

Prostaglandin E2 and Its Receptor EP2 Modulate Macrophage Activation and Fusion in Vitro.

The foreign body response (FBR) has impaired progress of new implantable medical devices through its hallmark of chronic inflammation and foreign body giant cell (FBGC) formation leading to fibrous encapsulation. Macrophages are known to drive the FBR, but efforts to control macrophage polarization remain challenging. The goal for this study was to investigate whether prostaglandin E2 (PGE2), and specifically its receptors EP2 and/or EP4, attenuate classically activated (i.e., inflammatory) macrophages and macrophage fusion into FBGCs in vitro. Lipopolysaccharide (LPS)-stimulated macrophages exhibited a dose-dependent decrease in gene expression and protein production of tumor necrosis factor alpha (TNF-α) when treated with PGE2. This attenuation was primarily by the EP4 receptor, as the addition of the EP2 antagonist PF 04418948 to PGE2-treated LPS-stimulated cells did not recover TNF-α production while the EP4 antagonist ONO AE3 208 did. However, direct stimulation of EP2 with the agonist butaprost to LPS-stimulated macrophages resulted in a ∼60% decrease in TNF-α secretion after 4 h and corresponded with an increase in gene expression for Cebpb and Il10, suggesting a polarization shift toward alternative activation through EP2 alone. Further, fusion of macrophages into FBGCs induced by interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) was inhibited by PGE2 via EP2 signaling and by an EP2 agonist, but not an EP4 agonist. The attenuation by PGE2 was confirmed to be primarily by the EP2 receptor. Mrc1, Dcstamp, and Retlna expressions increased upon IL-4/GM-CSF stimulation, but only Retnla expression with the EP2 agonist returned to levels that were not different from controls. This study identified that PGE2 attenuates classically activated macrophages and macrophage fusion through distinct EP receptors, while targeting EP2 is able to attenuate both. In summary, this study identified EP2 as a potential therapeutic target for reducing the FBR to biomaterials.

Siglec-F is induced by granulocyte-macrophage colony-stimulating factor and enhances interleukin-4-induced expression of arginase-1 in mouse macrophages.

Siglecs are cell surface lectins that recognize sialic acids and are primarily expressed in hematopoietic cells. Previous studies showed that some Siglecs regulate macrophage function. In the present study, we examined the induction and putative roles of mouse Siglec-F in bone-marrow-derived macrophages in mice. A quantitative RT-PCR analysis showed that the basal expression of Siglec-F was weak in bone-marrow-derived macrophages differentiated by macrophage colony-stimulating factor. However, a 24-hr stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced Siglec-F expression. GM-CSF also enhanced Siglec-F expression in thioglycollate-induced peritoneal macrophages. The inhibition of signal transducer and activator of transcription 5 (STAT5), but not that of phosphoinositide 3-kinase or mitogen-activated protein kinase kinase, significantly reduced the induction of Siglec-F. Interleukin-3, which uses a common β-chain shared with the GM-CSF receptor to stimulate the STAT5 pathway, also enhanced Siglec-F expression. The knockdown of Siglec-F by a specific small interfering RNA enhanced GM-CSF-induced STAT5 phosphorylation, suggesting that Siglec-F down-regulates its own expression upon prolonged GM-CSF stimulation. Furthermore, the knockdown of Siglec-F reduced the STAT6 phosphorylation and expression of arginase-1 in interleukin-4-stimulated macrophages. These results suggest that Siglec-F fine-tunes the immune responses of macrophages.

The role of granulocyte macrophage colony stimulating factor in hospitalized children with Mycoplasma pneumoniae pneumonia.

Inappropriate inflammatory response in children with M. pneumoniae infection might be associated with disease severity. The role of Granulocyte macrophage colony stimulating factor (GM-CSF) in hospitalized children with Mycoplasma pneumoniae pneumonia (MPP) has not been fully discussed. Clinical and laboratory data of a total 40 children with MPP were collected. GM-CSF and myeloperoxidase (MPO) were detected by ELISAs. Meanwhile, normal human bronchial epithelium was infected by M.pneumoniae and neutrophils were stimulated by GM-CSF to explore GM-CSF and MPO release in supernatant, respectively. Compared to control group, a significant increased percentage of neutrophils and decreased percentage of macrophages in bronchoalveolar lavage fluid of children with MPP was observed (P<0.05). Children with MPP had significantly higher levels of GM-CSF (P=0.0047) and MPO (P=0.0002) in BALF compared to the controls. Level of GM-CSF in BALF was associated with duration of fever (r=0.42, P=0.007) and strongly correlated with level of MPO (r=0.075, P=0.0005). Levels of GM-CSF and MPO significantly decreased (both P<0.05) after treatment. Invitro, M.pneumoniae induced GM-CSF expression in a time-dependent manner during a 72-h period (P<0.05) and MPO secretion significantly increased by recombinant human GM-CSF stimulation at 24h (P<0.05). GM-CSF could be induced by M.pneumoniae infection in vivo and vitro. Childen with high level GM-CSF had longer duration of fever. GM-CSF probably plays a vital role in neutrophil inflammation in M.pneumoniae infection.

Tolerogenic bone marrow-derived dendritic cells induce neuroprotective regulatory T cells in a model of Parkinson\u2019s disease

BackgroundAdministration of granulocyte-macrophage colony-stimulating factor (GM-CSF) increases regulatory T cell (Treg) number and function with control of neuroinflammation and neuronal protection in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson’s disease (PD). Recently, we demonstrated in an early phase 1 clinical trial that GM-CSF also improves motor skills in PD patients. However, the mechanisms of Treg induction and its effects on neuroprotective responses remain unknown. As GM-CSF induces tolerogenic dendritic cells (DCs) that in turn convert conventional T cells to Tregs, the pathways for DC induction of Tregs were assessed.MethodsFollowing differentiation, bone marrow-derived dendritic cells (BMDCs) were cultured in media or GM-CSF with or without post-culture stimulation with nitrated α-synuclein (N-α-Syn). Expression of cell surface co-stimulatory molecules and proinflammatory cytokines, and induction of Tregs were evaluated. The neuroprotective capacity of tolerogenic BMDCs was assessed by adoptive transfer to MPTP-intoxicated mice. The extent of neuroinflammation and numbers of surviving dopaminergic neurons were assessed in relation to Treg numbers.ResultsCo-culture of differentiated BMDCs with conventional T cells led to Treg induction. Stimulation of BMDCs with N-α-Syn increased expression of co-stimulatory molecules and proinflammatory cytokines, with modest increases in Treg numbers. In contrast, continued culture of BMDCs with GM-CSF modestly altered expression of co-stimulatory molecules and proinflammatory cytokines and chemokines, but decreased Treg induction. Continued culture in GM-CSF and combined stimulation with N-α-Syn reduced Treg induction to the lowest levels. Adoptive transfer of tolerogenic BMDCs to MPTP-intoxicated mice increased splenic Tregs, attenuated neuroinflammatory responses, and protected nigrostriatal dopaminergic neurons.ConclusionsGM-CSF acts broadly to differentiate DCs and affect immune transformation from effector to regulatory immune responses. DCs skew such immune responses by increasing Treg numbers and activities that serve to attenuate proinflammatory responses and augment neuroprotection.

Cellular Differentiation of Human Monocytes Is Regulated by Time-Dependent Interleukin-4 Signaling and the Transcriptional Regulator NCOR2

SummaryHuman in vitro generated monocyte-derived dendritic cells (moDCs) and macrophages are used clinically, e.g., to induce immunity against cancer. However, their physiological counterparts, ontogeny, transcriptional regulation, and heterogeneity remains largely unknown, hampering their clinical use. High-dimensional techniques were used to elucidate transcriptional, phenotypic, and functional differences between human in vivo and in vitro generated mononuclear phagocytes to facilitate their full potential in the clinic. We demonstrate that monocytes differentiated by macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulating factor (GM-CSF) resembled in vivo inflammatory macrophages, while moDCs resembled in vivo inflammatory DCs. Moreover, differentiated monocytes presented with profound transcriptomic, phenotypic, and functional differences. Monocytes integrated GM-CSF and IL-4 stimulation combinatorically and temporally, resulting in a mode- and time-dependent differentiation relying on NCOR2. Finally, moDCs are phenotypically heterogeneous and therefore necessitate the use of high-dimensional phenotyping to open new possibilities for better clinical tailoring of these cellular therapies.

An epithelial-to-mesenchymal transition-inducing potential of granulocyte macrophage colony-stimulating factor in colon cancer

Growing evidence shows that granulocyte macrophage colony-stimulating factor (GM-CSF) has progression-promoting potentials in certain solid tumors, which is largely attributed to the immunomodulatory function of this cytokine in tumor niches. However, little is known about the effect of GM-CSF on cancer cells. Herein, we show that chronic exposure of colon cancer cells to GM-CSF, which harbor its receptor, leads to occurrence of epithelial to mesenchymal transition (EMT), in time and dose-dependent manners. These GM-CSF-educated cancer cells exhibit enhanced ability of motility in vitro and in vivo. Furthermore, GM-CSF stimulation renders colon cancer cells more resistant to cytotoxic agents. Mechanistic investigation reveals that MAPK/ERK signaling and EMT-inducing transcription factor ZEB1 are critical to mediate these effects of GM-CSF. In specimen of CRC patients, high-level expression of GM-CSF positively correlates with local metastases in lymph nodes. Moreover, the co-expression of GM-CSF and its receptors as well as phosphorylated ERK1/2 are observed. Thus, our study for the first time identifies a progression-promoting function of GM-CSF in colon cancer by inducing EMT.

The Effects of Ex Vivo Administration of Granulocyte-Macrophage Colony-Stimulating Factor and Endotoxin on Cytokine Release of Whole Blood Are Determined by Priming Conditions.

Background Lipopolysaccharide- (LPS-) induced tumour necrosis factor alpha (TNFα) secretion in critically ill patients can be considered as a measure of immune responsiveness. It can be enhanced by granulocyte-macrophage colony stimulating factor (GM-CSF). We investigated the effect of GM-CSF on ex vivo stimulated cytokine production using various preincubation regimens in healthy donors and patients with sepsis. Results The maxima for the stimuli occurred 3 hours after stimulation. In donors, there was an increase (p < 0.001) of LPS-induced TNFα levels following incubation with GM-CSF. The simultaneous incubation with GM-CSF and LPS caused an inhibition of TNFα production (p < 0.001). Postincubation with GM-CSF did not yield any difference. In patients, preincubation with GM-CSF yielded an enhanced ex vivo TNFα-response when TNFα levels were low. Patients with increased TNFα concentrations did not show a GM-CSF stimulation effect. The GM-CSF preincubation yielded an increase of IL-8 production in patients and donors. Conclusions This study demonstrates the immune-modulating properties of GM-CSF depending on the absence or presence of LPS or systemic TNFα. The timing of GM-CSF administration may be relevant for the modulation of the immune system in sepsis. The lack of stimulation in patients with high TNFα may represent endotoxin tolerance.

Alarmin S100A9 Induces Proinflammatory and Catabolic Effects Predominantly in the M1 Macrophages of Human Osteoarthritic Synovium

The alarmins S100A8 and S100A9 have been shown to regulate synovial activation, cartilage damage, and osteophyte formation in osteoarthritis (OA). Here we investigated the effect of S100A9 on the production of proinflammatory cytokines and matrix metalloprotease (MMP) in OA synovium, granulocyte macrophage colony-stimulating factor (GM-CSF)-differentiated/macrophage colony-stimulating factor (M-CSF)-differentiated macrophages, and OA fibroblasts. We determined which cell types in the synovium produced S100A8 and S100A9. Further, the production of proinflammatory cytokines and MMP, and the activation of canonical Wnt signaling, was determined in human OA synovium, OA fibroblasts, and monocyte-derived macrophages following stimulation with S100A9. We observed that S100A8 and S100A9 were mainly produced by GM-CSF-differentiated macrophages present in the synovium, and to a lesser extent by M-CSF-differentiated macrophages, but not by fibroblasts. S100A9 stimulation of OA synovial tissue increased the production of the proinflammatory cytokines interleukin (IL) 1β, IL-6, IL-8, and tumor necrosis factor-α. Additionally, various MMP were upregulated after S100A9 stimulation. Experiments to determine which cell type was responsible for these effects revealed that mainly stimulation of GM-CSF-differentiated macrophages and to a lesser extent M-CSF-differentiated macrophages with S100A9 increased the expression of these proinflammatory cytokines and MMP. In contrast, stimulation of fibroblasts with S100A9 did not affect their expression. Finally, stimulation of GM-CSF-differentiated, but not M-CSF-differentiated macrophages with S100A9 activated canonical Wnt signaling, whereas incubation of OA synovium with the S100A9 inhibitor paquinimod reduced the activation of canonical Wnt signaling. Predominantly mediated by M1-like macrophages, the alarmin S100A9 stimulates the production of proinflammatory and catabolic mediators and activates canonical Wnt signaling in OA synovium.

GM-CSF Enhances Macrophage Glycolytic Activity In Vitro and Improves Detection of Inflammation In Vivo.

(18)F-FDG accumulates in glycolytically active tissues and is known to concentrate in tissues that are rich in activated macrophages. In this study, we tested the hypotheses that human granulocyte-macrophage colony-stimulating factor (GM-CSF), a clinically used cytokine, increases macrophage glycolysis and deoxyglucose uptake in vitro and acutely enhances (18)F-FDG uptake within inflamed tissues such as atherosclerotic plaques in vivo. In vitro experiments were conducted on human macrophages whereby inflammatory activation and uptake of radiolabeled 2-deoxyglucose was assessed before and after GM-CSF exposure. In vivo studies were performed on mice and New Zealand White rabbits to assess the effect of GM-CSF on (18)F-FDG uptake in normal versus inflamed arteries, using PET. Incubation of human macrophages with GM-CSF resulted in increased glycolysis and increased 2-deoxyglucose uptake (P < 0.05). This effect was attenuated by neutralizing antibodies against tumor necrosis factor-α or after silencing or inhibition of 6-phosphofructo-2-kinase. In vivo, in mice and in rabbits, intravenous GM-CSF administration resulted in a 70% and 73% increase (P < 0.01 for both), respectively, in arterial (18)F-FDG uptake in atherosclerotic animals but not in nonatherosclerotic controls. Histopathologic analysis demonstrated a significant correlation between in vivo (18)F-FDG uptake and macrophage staining (R = 0.75, P < 0.01). GM-CSF substantially augments glycolytic flux in vitro (via a mechanism dependent on ubiquitous type 6-phosphofructo-2-kinase and tumor necrosis factor-α) and increases (18)F-FDG uptake within inflamed atheroma in vivo. These findings demonstrate that GM-CSF can be used to enhance detection of inflammation. Further studies should explore the role of GM-CSF stimulation to enhance the detection of inflammatory foci in other disease states.

Flow cytometric analysis of intracellular phosphoproteins in human monocytes.

Using antibodies against intracellular phosphoproteins, flow cytometry can be used to monitor simultaneously multiple signaling pathways. Here, we tested a recently released procedure to analyze phosphorylation events in human monocytes upon different types of stimulation. Whole blood was treated by lipopolysaccharide (LPS) or granulocyte-macrophage colony-stimulating factor (GM-CSF), then cells were labeled by antibodies recognizing cell surface and cytosolic proteins. Human monocytes were identified by a CD14 - CD45 staining and three phosphorylated proteins such as AKT, ERK-1/2, and STAT5, were simultaneously detected by multicolor phosphoflow analysis. By this rapid method, we are able to detect directly from a blood sample several signaling events in human monocytes where LPS stimulation induces preferentially ERK-1/2 phosphorylation where as GM-CSF stimulation induces STAT5 phosphorylation. This procedure provides a simultaneous measurement of multiple activated signaling molecules using a simplified and rapid protocol. © 2015 International Clinical Cytometry Society.

Translational initiation regulated by ATM in dendritic cells development

Ataxia telangiectasia mutated (ATM) protein has been implicated in multiple pathways such as DNA repair, cell cycle checkpoint, cell growth, development, and stem cell renewal. In this study, we demonstrate evidence that ATM is involved in granulocyte macrophage colony-stimulating factor (GM-CSF)-induced dendritic cell (DC) development from bone marrow (BM) cells. Inactivation of ATM protein results in decreased BM proliferation, leading to reduced DC development and their activity for T cell activation. Expression of Jak2, STAT5, and mTOR is suppressed in both wild-type and ATM-null BM prior to GM-CSF stimulation. Activation of those proteins is delayed and prolonged hypophosphorylation of 4EBP1 is observed in ATM-null BM when treated with GM-CSF, although Erk and p38 are similarly expressed and activated in both wild-type and ATM-null BM cell types. Akt is also suppressed in wild-type BM, and transduction of constitutively active Akt or STAT5 in ATM-null BM restores DC development. Together, these results illustrate that ATM deficiency causes impaired initiation of protein translation in BM, leading to immature development of DC.

Nicotine inhibits microglial proliferation and is neuroprotective in global ischemia rats.

Ischemic injury in rodent models reliably leads to the activation of microglia, which might play a detrimental role in neuronal survival. Our preliminary studies suggest that nicotine plays a potential role in decreasing the numbers of cultured microglia in vitro. In the present study, we found treatment with nicotine 2, 6, and 12h after ischemia for 7days significantly increased the survival of CA1 pyramidal neurons in ischemia/reperfusion rats. This effect was accompanied by a significant reduction in the increase of microglia rather than astrocytes, as well as a significant reduction of enhanced expression of tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β) in CA1 induced by ischemia/reperfusion. Nicotine inhibits microglial proliferation in primary cultures with and without the stimulation of granulocyte-macrophage colony-stimulating factor (GM-CSF). Pre-treatment with α-bungarotoxin, a selective α7 nicotinic acetylcholine receptor (α7 nAChR) antagonist, could prevent the inhibitory effects of nicotine on cultured microglial proliferation suggesting that nicotine inhibits the microglial proliferation in an α7 nAChR-dependent fashion. Our results suggest that nicotine inhibits the inflammation mediated by microglia via α7 nAChR and is neuroprotective against ischemic stroke, even when administered 12h after the insult. α7 nAChR agonists may have uses as anti-ischemic compounds in humans.

Juvenile myelomonocytic leukaemia-associated mutation in Cbl promotes resistance to apoptosis via the Lyn-PI3K/AKT pathway

Juvenile myelomonocytic leukaemia (JMML) is an aggressive myeloproliferative neoplasm in children characterized by granulocyte macrophage colony-stimulating factor (GM-CSF) hypersensitivity and resistance to chemotherapy. We recently identified c-Cbl (henceforth referred to as Cbl) as a GM-CSF receptor (GMR) responsive protein that targets Src for ubiquitin-mediated destruction upon GM-CSF stimulation and showed that a loss of negative regulation of Src is pivotal in the hyperactivation of GMR signalling in JMML cells. However, the mechanism regulating the chemoresistant nature of JMML has remained largely unknown. Here, we show that the JMML-associated Cbl mutant in complex with the Src family kinase Lyn promotes Cbl's adapter function, leading to increased association to PI3K regulatory subunit p85 and Lyn-dependent AKT pro-survival signalling. Notably, molecular or pharmacologic inhibition of the Lyn-PI3K/AKT pathway, but not the Ras/mitogen-activated protein kinase signalling axis, markedly increased the sensitivity of the otherwise chemoresistant Cbl mutant-JMML cells to chemotherapeutic agents currently used in the treatment of JMML patients. These results support the potential translational benefit of combining modalities that inhibit Lyn-PI3K/AKT signalling with traditional antileukaemia agents in the management of JMML.