Granulocyte-macrophage Colony-stimulating Factor Receptor Expression Research Articles

GM-CSF receptor expression determines opposing innate memory phenotypes at different stages of myelopoiesis

AbstractInflammatory responses must be tightly coordinated with the activation of emergency myelopoiesis to produce potent myeloid cells that fight infection without causing excessive host damage. Here, we show that granulocyte-macrophage colony-stimulating factor (GM-CSF) programs myeloid-committed progenitors to produce trained macrophages (increased cytokine response), but programs the upstream noncommitted LKS+ progenitors (defined as Lin− c-Kit+ Sca-1+ cells) to produce tolerized macrophages (decreased cytokine response). In myeloid progenitors, GM-CSF strongly activates signal transducer and activator of transcription 5 (STAT5), Ras-Raf-extracellular signal regulated kinase (ERK), and Akt-mTOR signaling pathways, which are essential to establish a training program, whereas in LKS+ progenitors, GM-CSF induces NF-κB translocation to the nucleus to establish a tolerization program. These differences arise from higher GM-CSF receptor expression in myeloid progenitors compared with LKS+ cells. We demonstrate that β-catenin regulation of NF-κB nuclear translocation is central in this process. In myeloid progenitors, glycogen synthase kinase 3 (GSK3) inactivation by strong ERK and phosphatidylinositol 3 kinase (PI3K)-Akt signaling increases cytoplasmic β-catenin levels to block NF-κB nuclear translocation. In contrast, when ERK and PI3K-Akt signaling are weak, active GSK3 causes a decrease in β-catenin, allowing NF-κB nuclear translocation in LKS+ progenitors. Finally, GM-CSF-induced LKS+ tolerization takes place in several murine models of trained immunity and in human CD34+ CD38− progenitors. Our study reveals that in addition to activating myelopoiesis, GM-CSF also programs early and immediate myeloid progenitors to produce opposing immune memory phenotypes. We propose that the inflammatory response from immediate myeloid progenitors may be balanced by the tolerized phenotype of early progenitors, thus providing a mechanism for appropriate resolution of inflammation and protection against a prolonged cytokine storm.

Granulocyte-macrophage colony-stimulating factor: Conductor of the wound healing orchestra?

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein and is derived from both hemopoietic and nonhemopoietic sources which exert immunomodulatory properties. Various theories have been proposed to explain why some wounds become chronic and non-healing. Generalized suppression of inflammation locally or systemically may impede the body's physiological healing response by crippling the activity of reparative cells within the wound ecosystem. Thus, highlighting the importance of promoting host-directed therapeutics with immunomodulatory properties. The temporal and spatial expression of GM-CSF and GM-CSF receptors in the integumentary system suggests that epithelial-derived GM-CSF functions in an autocrine/paracrine manner. This may positively affect wound healing physiology via local inflammatory regulation promoting macrophage survival. Although diabetes negatively affects multiple aspects of wound healing GM-CSF activation is particularly impacted. Compared to acute/healthy wounds diabetic foot ulcers (DFU) only partially activate GM-CSF activity. There is a deleterious chain of events associated with this unfortunate sequala. DFUs also have a high proportion of monocytes and an absence of activated macrophages which results in an impaired inflammatory response. This may potentially serve as a vital point for GM-CSF to act as a companion diagnostic/theragnostic modality to help modulate the inflammatory response in wound healing. Correcting macrophage immune dysfunction with exogenous GM-CSF may help restore the immune balance in the wound ecosystem and jumpstart the wound healing cascade. Thus, the recognized beneficial role of GM-CSF in immune regulation across many studies provides a rationale for the initiation of the ongoing randomized controlled trials using GM-CSF.

The Role of Zinc in GM-CSF-Induced Signaling in Human Polymorphonuclear Leukocytes.

Zinc is suggested to be necessary for functional signaling induced by certain growth factors. The granulocyte-macrophage colony-stimulating factor (GM-CSF) is a key factor for differentiation and activation of myeloid cells. This report analyses the impact of different zinc concentrations on GM-CSF-induced signaling in mature polymorphonuclear leukocytes (PMN). As measured by flow cytometry, zinc increases surface GM-CSF receptor (GM-CSFR) in PMN, whereas monocytes respond with decreased GM-CSFR surface expression. Since total cellular GM-CSFR expression remains unaffected, the observed zinc-induced GM-CSFR surface dynamics may be explained by receptor redistribution. In PMN, zinc enhanced phosphorylation of mitogen-activated protein kinases (MAPK) in a dose-dependent manner as found in western blot. Zinc-induced MAPK phosphorylation is additionally augmented by moderate GM-CSF stimulation. The present study demonstrates the opposing influence of zinc on GM-CSFR surface expression in monocytes and PMN. Zinc and GM-CSF, use in optimized concentrations, augment MAPK signaling, and increase expression of MAPK-induced myeloid cell leukemia-1 (Mcl-1) in PMN. Thus, this study concludes that zinc strengthens growth factor-induced signaling. Hence, the study provides a basis for further in vivo studies, focusing on the therapeutic value of zinc in patients with a disturbed GM-CSF signaling.

Granulocyte Macrophage-Colony Stimulating Factor Produces a Splenic Subset of Monocyte-Derived Dendritic Cells That Efficiently Polarize T Helper Type 2 Cells in Response to Blood-Borne Antigen.

Dendritic cells (DCs) are key antigen-presenting cells that prime naive T cells and initiate adaptive immunity. Although the genetic deficiency and transgenic overexpression of granulocyte macrophage-colony stimulating factor (GM-CSF) signaling were reported to influence the homeostasis of DCs, the in vivo development of DC subsets following injection of GM-CSF has not been analyzed in detail. Among the treatment of mice with different hematopoietic cytokines, only GM-CSF generates a distinct subset of XCR1-33D1- DCs which make up the majority of DCs in the spleen after three daily injections. These GM-CSF-induced DCs (GMiDCs) are distinguished from classical DCs (cDCs) in the spleen by their expression of CD115 and CD301b and by their superior ability to present blood-borne antigen and thus to stimulate CD4+ T cells. Unlike cDCs in the spleen, GMiDCs are exceptionally effective to polarize and expand T helper type 2 (Th2) cells and able to induce allergic sensitization in response to blood-borne antigen. Single-cell RNA sequencing analysis and adoptive cell transfer assay reveal the sequential differentiation of classical monocytes into pre-GMiDCs and GMiDCs. Interestingly, mixed bone marrow chimeric mice of Csf2rb +/+ and Csf2rb -/- demonstrate that the generation of GMiDCs necessitates the cis expression of GM-CSF receptor. Besides the spleen, GMiDCs are generated in the CCR7-independent resident DCs of the LNs and in some peripheral tissues with GM-CSF treatment. Also, small but significant numbers of GMiDCs are generated in the spleen and other tissues during chronic allergic inflammation. Collectively, our present study identifies a splenic subset of CD115hiCD301b+ GMiDCs that possess a strong capacity to promote Th2 polarization and allergic sensitization against blood-borne antigen.

Attenuated Hematopoietic Response to Granulocyte-Macrophage Colony-Stimulating Factor in Patients With Acquired Pulmonary Alveolar Proteinosis

The pathogenesis of acquired pulmonary alveolar proteinosis (PAP), a rare lung disease characterized by excessive surfactant accumulation within the alveolar space, remains obscure. Gene-targeted mice lacking the hematopoietic growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF) or the signal-transducing β-common chain of the GM-CSF receptor have impaired surfactant clearance and pulmonary pathology resembling human PAP. We therefore investigated the hematopoietic effects of GM-CSF in patients with PAP. The hematologic response of 5 infants with congenital PAP to 5 μg/kg/d was of normal magnitude. By contrast, despite normal expression of GM-CSF receptor α- and β-common chains on peripheral blood myelomonocytic cells (n = 6) and normal binding affinity of bone marrow mononuclear cells for GM-CSF (n = 3), each of the 12 patients with acquired PAP treated displayed impaired responses to GM-CSF; 5 μg/kg/d produced only minor eosinophilia, and doses of 7.5 to 20 μg/kg were required to induce ≥1.5-fold neutrophil increments in the 3 patients who underwent dose-escalation. However, neutrophilic responses to 5 μg/kg granulocyte colony-stimulating factor (G-CSF) were normal (n = 4). In vitro, the proportion of hematopoietic progenitors responsive to GM-CSF (16.1% ± 8.9%; P = .042) or interleukin-3 (IL-3; 19.3% ± 7.7%; P = .063), both of which utilize the β-common chain of the GM-CSF receptor complex, were reduced among patients with acquired PAP (n = 4) compared with normal bone marrow donor controls (47.2% ± 25.9% and 40.9% ± 18.6%, respectively). In the one individual who had complete resolution of lung disease during the period of study, this was temporally associated with correction of this defective in vitro response to GM-CSF and IL-3 on serial assessment. These data establish that patients with acquired PAP have an associated impaired responsiveness to GM-CSF that is potentially pathogenic in the development of their lung disease. Based on these observations, we propose a model of the pathogenesis of acquired PAP that suggests the disease arises as a consequence of an acquired clonal disorder within the hematopoietic progenitor cell compartment.© 1998 by The American Society of Hematology.

Granulocyte Macrophage-Colony Stimulating Factor Reverses HIV Protein-Induced Mitochondrial Derangements in Alveolar Macrophages.

Despite the advent of antiretroviral therapy, people living with HIV suffer from a range of infectious and noninfectious pulmonary complications. HIV impairs antioxidant defenses and innate immune function of the alveolar macrophage by diminishing granulocyte macrophage-colony stimulating factor (GM-CSF) signaling. Since GM-CSF may be linked to mitochondria, we sought to determine the effects of HIV on GM-CSF receptor expression and alveolar macrophage mitochondrial function. At an academic medical center, studies were completed on alveolar macrophages isolated from both wild-type and HIV transgenic (HIV Tg) rats and human subjects with and without HIV. Primary macrophages were plated and evaluated for expression of GM-CSF receptor beta, phagocytic index, and mitochondrial function in the presence and absence of GM-CSF treatment. GM-CSF receptor expression and mitochondrial function were impaired in macrophages isolated from HIV Tg rats, and treatment with GM-CSF restored GM-CSF receptor expression and mitochondrial function. GM-CSF treatment of HIV Tg rats also increased alveolar macrophage levels of the mitochondrial proteins voltage-dependent anion-selective channel 1 (VDAC) and glucose-regulated protein 75 (Grp75). Similar to the HIV Tg rat model, impairments in mitochondrial bioenergetics were confirmed in alveolar macrophages isolated from human subjects with HIV. HIV-associated impairments in alveolar macrophage mitochondrial bioenergetics likely contribute to innate immune dysfunction in HIV infection, and GM-CSF treatment may offer a novel therapeutic strategy for mitigating these deleterious effects.

Improved Anti-Tumor Response of Chimeric Antigen Receptor T Cell (CART) Therapy after GM-CSF Inhibition Is Mechanistically Supported By a Novel Direct Interaction of GM-CSF with Activated Carts

Despite the remarkable activity of CD19 directed chimeric antigen receptor T cell (CART19) therapy in the treatment of B cell malignancies, the therapy is limited by the development of severe life-threatening complications such as neurotoxicity (NT) and cytokine release syndrome (CRS). Additionally, durable efficacy following CART19 therapy is not optimal. Emerging literature suggests that inhibitory myeloid cells and their cytokines play an important role in inducing CAR-T cell toxicities and also contribute to the inhibition of their effector functions. Specifically, GM-CSF was identified as a critical cytokine in the development of NT and CRS after CART19 therapy. Neutralization of GM-CSF in preclinical models has been shown to prevent CRS and enhance CART anti-tumor activity through modulation of myeloid cell behavior, resulting in reduction of tumor associated macrophages. In addition to the predominant effect of GM-CSF on myeloid cells, there appears to be a direct effect on CART19 cells. In this study, we aimed to evaluate the direct effect of GM-CSF on CART cells. Our initial finding of enhanced anti-tumor activity of CART19 cells after GM-CSF inhibition suggested a direct effect of GM-CSF on CART cells (Sterner et al. 2019, Blood). In these experiments, a guide RNA (gRNA) targeting exon 3 of GM-CSF in a CRISPR-Cas9 lentiviral vector was used to knock out GM-CSF during CART cell manufacturing. This resulted in a disruption efficiency of approximately 70% of the GM-CSF gene. Using a high tumor burden xenograft model for relapsed acute lymphoblastic leukemia established through the engraftment of the CD19+ luciferase+ NALM6 cell line (1x106 cells intravenously) in immunocompromised NOD-SCID-γ-/- mice, treatment with low doses of GM-CSFk/o CART19 resulted in improved anti-tumor activity and overall survival compared to GM-CSFwt CART19. The lack of myeloid cells in this model pointed to an intrinsic effect of GM-CSF on CAR-T cells. To ensure that this was not related to an off-target effect of the gRNA, whole exome sequencing (WES) of the modified cells was performed. There was no difference in the single nucleotide variants or indel counts between GM-CSFk/o CART19 and GM-CSFwt CART19 (Figure 1A). WES was significant for only two alterations in the exon 3 targeted by the gRNA (Figure 1B). The high efficiency and accuracy of targeting exon 3 of GM-CSF indicated that the improvement in CART function is unlikely related to an off-target effect of the gRNA and suggested the possibility of a direct interaction between GM-CSF and CART cells as a potential mechanism behind the improved anti-tumor activity. To investigate this interaction, we first assessed the expression of GM-CSF receptors on CART cells. While resting CART cells do not express any GM-CSF receptors, our analysis robustly indicates that activated CART cells significantly upregulate both α and β subunits of the GM-CSF receptor. This finding was significant both when CART cells are activated through their T cell receptor with CD3/CD28 beads (Figure 1C) or through the CAR with irradiated NALM6 cells (Figure 1D). Additionally, activated GM-CSFk/o CART19 cells also upregulated GM-CSF receptors, indicating this upregulation is induced by T cell stimulation. These results suggest a direct interaction between GM-CSF and upregulated GM-CSFR on activated CART cells. Having demonstrated that 1) GM-CSF depletion enhances CART19 efficacy in xenograft models in the absence of monocytes and 2) T cell activation increases GM-CSF receptor expression, we sought to uncover the downstream changes resulting from this effect. Transcriptome interrogation of GM-CSFk/o CART19 revealed a distinct signature including a significant inhibition of the Fas death pathway, a known critical pathway in inducing CART cell apoptosis. This suggests a potential mechanism for enhanced CART19 activity following GM-CSF depletion (Figure 1E). In summary, our results strongly indicate that CART cells increase expression of GM-CSF receptor subunits when activated, resulting in modulation of CART cell functions. Furthermore, GM-CSFk/o CART19 revealed a distinct transcriptome signature compared to GM-CSFwt CART19. These results illuminate a novel mechanism for a direct modulatory effect of GM-CSF on activated CART cells. Disclosures Cox: Humanigen: Patents & Royalties. Sterner:Humanigen: Patents & Royalties. Sakemura:Humanigen: Patents & Royalties. Ahmed:Humanigen: Employment. Chappell:Humanigen: Employment. Durrant:Humanigen: Employment. Parikh:Acerta Pharma: Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Genentech: Honoraria; Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; Ascentage Pharma: Research Funding. Kay:MorphoSys: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Agios: Other: DSMB. Kenderian:Novartis: Patents & Royalties, Research Funding; Tolero: Research Funding; Lentigen: Research Funding; Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Kite/Gilead: Research Funding; Morphosys: Research Funding.

Role of GM-CSF in a mouse model of experimental autoimmune prostatitis.

The etiology of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is still unknown. Granulocyte macrophage colony-stimulating factor (GM-CSF) has been shown to play an important role in the development of autoimmune and inflammatory diseases. Here, we investigated the expression and function of GM-CSF in patients with CP/CPPS and in a mouse model of experimental autoimmune prostatitis (EAP). GM-CSF mRNA levels were detected in expressed prostatic secretions samples from patients with CP/CPPS and in prostate tissue from a mouse model of EAP. The expression of GM-CSF receptor in mouse prostate and dorsal root ganglia were determined using PCR and immunohistochemistry. Behavioral testing and inflammation scoring were performed to evaluate the role of GM-CSF in disease development and symptom severity of EAP using GM-CSF knockout mice. mRNA levels of putative nociceptive and inflammatory markers were measured in the prostate after the induction of EAP. Elevated GM-CSF mRNA levels were observed in expressed prostatic secretions samples from patients with CP/CPPS compared with healthy volunteers. GM-CSF mRNA was also significantly increased in prostate tissue of the EAP mice model. The expression of GM-CSF receptors was confirmed in mouse prostate and dorsal root ganglia. GM-CSF knockout mice showed fewer Infiltrating leukocytes and pain symptoms after the induction of EAP. Deletion of GM-CSF significantly diminished EAP-induced increases of chemokine (C-C motif) ligand 2, chemokine (C-C motif) ligand 3, and nerve growth factor mRNA expression. The results indicated that GM-CSF plays a functional role in the pathogenesis of EAP. GM-CSF may function as a signaling mediator for both inflammation and pain transduction in CP/CPPS.

The Relationship Between Cytokine Production, CSF2RA, and IL1R2 Expression in Mammary Adenocarcinoma, Tumor Histopathological Parameters, and Lymph Node Metastasis

Objective:The aim of this study was to evaluate the relationship between cytokine production, GM-CSF receptor (CSF2RA), and IL-1 receptor (IL1R2) expression in mammary adenocarcinoma and their association with it histopathological parameters and lymph node metastasis.Methods:We analyzed tumor biopsy samples (cultured in vitro) from 50 women (aged 43-75) with invasive ductal mammary adenocarcinomas. Enzyme-linked immunosorbent assay method the concentrations of interleukin 2, interleukin 6, interleukin 8, interleukin 10, interleukin 17, interleukin 18, interleukin 1β, interleukin 1Ra, tumor necrosis factor α, interferon γ, granulocyte colony-stimulating factor, granulocyte macrophage colony-stimulating factor, and vascular endothelial growth factor A were determined in culture supernatants. The expression of CSF2RA and IL1R2 in tumor biopsy was evaluated by immunohistochemical method.Results:We showed that the “cytokine profile” of a tumor (the ability of tumor cells and its microenvironment to produce different cytokines) is very individual. It has been shown that the features of the cytokine profile of the mammary adenocarcinoma are important for the formation and realization of the metastatic potential of the mammary adenocarcinoma. We found correlations between some histopathological parameters of mammary adenocarcinoma and coefficients KGM-CSF/CSF2RA and KIL-1β/IL1R2, which are the ratios of concentrations of granulocyte macrophage colony-stimulating factor and interleukin -1β to expression of CSF2RA and IL1R2, respectively. KGM-CSF/CSF2RA positively correlated with highly differentiated cells, and KIL-1β/IL1R2 positively correlated with the number of mitoses, poorly differentiated cells, and a number of lymph nodes with metastases. KGM-CSF/CSF2RA positively correlated with the concentrations of interleukin 6, interleukin 8, interleukin 1Ra, and granulocyte colony-stimulating factor. KIL-1β/IL1R2 positively correlated with concentrations of interleukin 1β and interferon γ and negative correlated with the concentrations of vascular endothelial growth factor A and tumor necrosis factor α. It is shown that KIL-1β/IL1R2 can be considered as a prognostic indicator predicting the probability of mammary adenocarcinoma metastasis to regional lymph nodes.Conclusions:The ratios of granulocyte macrophage colony-stimulating factor and interleukin 1β cytokines, produced in tumor, to the expression of CSF2RA and IL1R2 depend on levels of interleukin 6, interleukin 8, tumor necrosis factor α, interferon γ, granulocyte colony-stimulating factor, and vascular endothelial growth factor A and are important factors affecting the progression and metastasis of the breast cancer.

Granulocyte-macrophage colony-stimulating factor receptor expression in clinical pain disorder tissues and role in neuronal sensitization.

Granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) is highly expressed in peripheral macrophages and microglia, and is involved in arthritis and cancer pain in animal models. However, there is limited information on GM-CSFR expression in human central nervous system (CNS), peripheral nerves, or dorsal root ganglia (DRG), particularly in chronic pain conditions. Immunohistochemistry was used to quantify GM-CSFR expression levels in human tissues, and functional sensory effects of GM-CSF were studied in cultured DRG neurons. Granulocyte-macrophage colony-stimulating factor receptor was markedly increased in microglia at lesional sites of multiple sclerosis spinal cords (P = 0.01), which co-localised with macrophage marker CD68 (P = 0.009). In human DRG, GM-CSFR was expressed in a subset of small/medium diameter cells (30%) and few large cells (10%), with no significant change in avulsion-injured DRG. In peripheral nerves, there was a marked decrease in axonal GM-CSFR after chronic painful nerve injury (P = 0.004) and in painful neuromas (P = 0.0043); CD-68-positive macrophages were increased (P = 0.017) but did not appear to express GM-CSFR. Although control synovium showed absent GM-CSFR immunostaining, this was markedly increased in macrophages of painful osteoarthritis knee synovium. Granulocyte-macrophage colony-stimulating factor receptor was expressed in 17 ± 1.7% of small-/medium-sized cultured adult rat DRG neurons, and in 27 ± 3.3% of TRPV1-positive neurons. Granulocyte-macrophage colony-stimulating factor treatment sensitized capsaicin responses in vitro, which were diminished by p38 MAPK or TrkA inhibitors. Our findings support GM-CSFR as a therapeutic target for pain and hypersensitivity in clinical CNS and peripheral inflammatory conditions. Although GM-CSFR was decreased in chronic painful injured peripheral nerves, it could mediate CNS neuroinflammatory effects, which deserves study.

Expression and role of granulocyte macrophage colony-stimulating factor receptor (GM-CSFR) and granulocyte colony-stimulating factor receptor (G-CSFR) on Ph-positive acute B lymphoblastic leukemia

ABSTRACTObjective: We observed that ph + ALL patients administrated with recombinant human G-CSF (rhG-CSF) after intense chemotherapy have presented a trend of disease relapse. Thus, we aim to thoroughly investigate the expression and role of GM-CSFR and G-CSFR on ph + ALL patients.Method: SUP-B15, BALL-1 and primary leukemia cells were used in this study. Transcript levels were analyzed by quantitative PCR while cell viability was measured using a CCK-8 assay. Flow cytometry was used to assess the different stages of cell cycle.Results: We found that the mRNA expression levels of GM-CSFR and G-CSFR were higher in patients with ph + ALL, as well as in SUP-B15 cells. rhG-CSF was also observed to promote the viability of SUP-B15 cells while inversely inhibiting BALL-1 cell viability. In addition, we also determined that rhG-CSF (100 ng/ml) decreased the sensitivity of SUP-B15 cells to imatinib and nilotinib, while the results were exactly the contrary for dasatinib.Conclusion: We demonstrated high expression levels of GM-CSFR and G-CSFR, as well as their promotable role for viability in ph + ALL cells. We further found that rhG-CSF influenced the sensitivity of SUP-B15 cells to TKIs.

Central inhibition of granulocyte-macrophage colony-stimulating factor is analgesic in experimental neuropathic pain.

With less than 50% of patients responding to the current standard of care and poor efficacy and selectivity of current treatments, neuropathic pain continues to be an area of considerable unmet medical need. Biological therapeutics such as monoclonal antibodies (mAbs) provide better intrinsic selectivity; however, delivery to the central nervous system (CNS) remains a challenge. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is well described in inflammation-induced pain, and early-phase clinical trials evaluating its antagonism have exemplified its importance as a peripheral pain target. Here, we investigate the role of this cytokine in a murine model of traumatic nerve injury and show that deletion of the GM-CSF receptor or treatment with an antagonizing mAb alleviates pain. We also demonstrate enhanced analgesic efficacy using an engineered construct that has greater capacity to penetrate the CNS. Despite observing GM-CSF receptor expression in microglia and astrocytes, the gliosis response in the dorsal horn was not altered in nerve injured knockout mice compared with wild-type littermate controls as evaluated by ionized calcium binding adapter molecule 1 (Iba1) and glial fibrillary acidic protein, respectively. Functional analysis of glial cells revealed that pretreatment with GM-CSF potentiated lipopolysaccharide-induced release of proinflammatory cytokines. In summary, our data indicate that GM-CSF is a proinflammatory cytokine that contributes to nociceptive signalling through driving spinal glial cell secretion of proinflammatory mediators. In addition, we report a successful approach to accessing CNS pain targets, providing promise for central compartment delivery of analgesics.

Serum amyloid A inhibits dendritic cell differentiation by suppressing GM-CSF receptor expression and signaling

In this study, we report that an acute phase reactant, serum amyloid A (SAA), strongly inhibits dendritic cell differentiation induced by GM-CSF plus IL-4. SAA markedly decreased the expression of MHCII and CD11c. Moreover, SAA decreased cell surface GM-CSF receptor expression. SAA also decreased the expression of PU.1 and C/EBPα, which play roles in the expression of GM-CSF receptor. This inhibitory response by SAA is partly mediated by the well-known SAA receptors, Toll-like receptor 2 and formyl peptide receptor 2. Taken together, we suggest a novel insight into the inhibitory role of SAA in dendritic cell differentiation.

Indispensable role of IL-10 in the development of antigen cross-presentation capacity of the cDC1 subset

Abstract The conventional type 1 dendritic cell (cDC1) is a distinct DC subset with specialized functions including cross-presentation of antigens to CD8 T cells and CD103 expression. cDC1s require GM-CSF signal for gaining their signature functions; however, the involvement of other cytokines remains unknown. Here we found that cDC1s were normal in number but lacked CD103 expression in IL-10-deficient (Il10−/−) mice, suggesting that IL-10 has some effects on cDC1 function in vivo. Thus, next we assessed the CD8 T-cell priming capacity of cDC1s from Il10+/+ and Il10−/− mice by coculture with OT-I CD8+ T cells in the presence of ovalbumin in vitro. In contrast to Il10+/+ cDC1s, Il10−/− cDC1s failed to induce the expansion and IFN-γ production of OT-I cells. In addition, Il10−/− mice had a significantly lower number of memory CD8 T cells in the lymph nodes in the steady state. These results indicate that cDC1s in Il10−/− mice were defective in antigen cross-presentation. We also found that, whereas Flt3-ligand-induced DCs derived from Il10+/+ bone marrow cells upregulated CD103 expression by and CD8 T-cell cross-priming capacity of the cDC1-equivalent population in response to GM-CSF stimulation, Il10−/− bone marrow-derived DCs did not, suggesting that dysfunction of cDC1s in Il10−/− mice is likely due to unresponsiveness to GM-CSF. Finally, we found that mRNA expression of Csf2ra, which encodes GM-CSF receptor α-chain, was completely deficient in both cDC1s in Il10−/− mice and Il10−/− cDC1-equivalent bone marrow-derived DCs. Collectively, our data suggest that IL-10 critically controls the development of antigen cross-presentation capacity of cDC1s under homeostatic conditions, likely through regulating GM-CSF receptor expression.

Fusion cytokine IL-2-GMCSF enhances anticancer immune responses through promoting cell-cell interactions.

Background Potent antitumor responses can be induced through cytokine immunotherapy. Interleukin (IL)-2 and granulocyte–macrophage colony-stimulating factor (GM-CSF) are among the most effective cytokines to induce tumor-specific systemic immune responses and can act synergistically. To overcome the limitations of combined use of these two cytokines, we have constructed an IL2-GMCSF fusion protein and characterized its antitumor effects in this study.MethodsThe expression of IL-2 receptor and GM-CSF receptor of cell lines were detected with quantitative real-time PCR. On this basis, the bioactivities of IL2-GMCSF, especially effects on DC2.4 cells were assayed. Another function of IL2-GMCSF—bridge two types of cells—was assessed by cell contact counting and cytotoxicity assays. The anti-tumor activity in vivo of IL2-GMCSF was evaluated in the melanoma model. The statistical significance among treatment groups were determined by One-Way ANOVA.ResultsThe fusion protein IL2-GMCSF maintained the activities of IL-2 and GM-CSF, and could significantly promote DC2.4 cell activities, including phagocytosis, proliferation and cytokine secretion. In addition to the inherent cytokine activity, IL2-GMCSF bridges direct cell–cell interactions and enhances splenocyte killing efficacy against multiple tumor cell lines in vitro. Co-injection of IL2-GMCSF and inactivated B16F10 mouse melanoma cells induced complete immunoprotective responses in about 30 % of mice.ConclusionThese results suggested that IL2-GMCSF can efficiently regulate immune responses against tumors. Furthermore, as the bridging effect relies on both IL-2R and GM-CSFR and promotes interactions between immune and tumor cells, IL2-GMCSF may be utilized as a useful tool for dissecting specific immune responses for future clinical applications.

The Toll-like receptor 4-activated neuroprotective microglia subpopulation survives via granulocyte macrophage colony-stimulating factor and JAK2/STAT5 signaling

Toll-like receptor (TLR) 4 mediates inflammation and is also known to trigger apoptosis in microglia. Our time-lapse observations showed that lipopolysaccharide (LPS) stimulation induced rapid death in primary cultures of rat microglia, while a portion of the microglia escaped from death and survived for much longer than 2 days, in which time, all of the control cells had died. However, it remains unclear how the LPS-stimulated microglia subpopulation could continue to survive in the absence of any supplied growth factors. In the present study, to clarify the mechanism underlying the LPS-stimulated survival, we investigated whether microglia could produce their own survival factors in response to LPS, focusing on macrophage colony-stimulating factor (M-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-34, which are mainly supplied by astrocytes or neurons. The LPS-stimulated microglia drastically induced the expression of the GM-CSF mRNA and protein, while M-CSF and IL-34 levels were unchanged. The surviving microglia also significantly upregulated the expression of GM-CSF receptor (GM-CSFR) mRNA without affecting M-CSFR. As for the GM-CSFR downstream signal, LPS resulted in the phosphorylation of STAT5 and its translocation to the nucleus in the surviving microglia. Moreover, a specific JAK2 inhibitor, NVP-BSK805, suppressed STAT5 phosphorylation and microglia survival in response to LPS, indicating a critical role of the JAK2/STAT5 pathway in this survival mechanism. Together, these results suggest that a subpopulation of TLR4-activated microglia may survive by producing GM-CSF and up-regulating GM-CSFR. This autocrine GM-CSF pathway may activate the JAK2/STAT5 signaling pathway, which controls the transcription of survival-related genes. Finally, these surviving microglia may have neuroprotective functions because the neurons remained viable in co-cultures with these microglia.

Granulocyte macrophage colony-stimulating factor treatment results in recovery of motor function after white matter damage in mice.

Clinical stroke usually results from a cerebral ischaemic event, and is frequently a debilitating condition with limited treatment options. A significant proportion of clinical strokes result from specific damage to the subcortical white matter (SWM), but currently there are few animal models available to investigate the pathogenesis and potential therapeutic strategies to promote recovery. Granulocyte macrophage colony-stimulating factor (GM-CSF) is a cytokine that has been previously shown to promote neuroprotective effects after brain damage; however, the mechanisms mediating this effect are not known. Here, it is reported that GM-CSF treatment results in dramatic functional improvement in a white matter model of stroke in mice. SWM stroke was induced in mice by unilateral injections of the vasoconstrictor, endothelin-1 (ET-1). The results reveal that ET-1-induced stroke impairs skilled motor function on the single pellet-reaching task and results in forelimb asymmetry, in adult mice. Treatment with GM-CSF, after stroke, restores motor function and abolishes forelimb asymmetry. The results also indicate that GM-CSF promotes its effects by activating mammalian target of rapamycin signalling mechanisms in the brain following stroke injury. Additionally, a significant increase in GM-CSF receptor expression was found in the ipsilateral hemisphere of the ET-1-injected brain. Taken together, the present study highlights the use of an under-utilized mouse model of stroke (using ET-1) and suggests that GM-CSF treatment can attenuate ET-1-induced functional deficits.

Differentiation, apoptosis, and GM-CSF receptor expression of human gingival fibroblasts on a titanium surface treated by a dual acid-etched procedure.

Analysis of the effects of titanium surface properties on the biological behavior of human gingival fibroblasts (HGFs). HGFs were in vitro cultured on a titanium surface modified by a dual acid-etched procedure and on a control machined surface. Cell adhesion, proliferation, apoptosis, production of certain extracellular matrix (ECM) proteins, and expression of granulocyte macrophage-colony stimulating factor receptor (GM-CSFR) were investigated using in each experiment a total of 18 samples for each titanium surface. Cell attachment at 3 h of culture was statistically significantly higher on the etched surface. HGF growth increased on both surfaces during the entire experimental period and at day 14 of culture cell proliferation was statistically significantly higher on the treated surface than on the control. No statistically significant differences in percentage of apoptosis events were observed between the surfaces. ECM protein production increased progressively over time on both surfaces. A statistically significant deposition was observed at day 7 and 14 for collagen I and only at day 14 for fibronectin and tenascin, when compared to the baseline. GM-CSFR registered a positive expression on both surfaces, statistically significant at day 14 on the etched surface in comparison with the machined one. Data showed that titanium surface microtopography modulates in vitro cell response and phenotypical expression of HGFs. The etched surface promoted a higher cell proliferation and differentiation improving the biological behavior of HGFs. Results suggest a possible beneficial effect of surface etching modification on peri-implant biological integration and soft tissue healing which is critical for the formation of a biological seal around the neck of dental implants.

GM-CSF-DFF40: a novel humanized immunotoxin induces apoptosis in acute myeloid leukemia cells

DNA fragmentation factor 40 (DFF40) is an endonuclease that acts downstream in the apoptotic cascade. The objective of this study was to generate a novel humanized chimeric protein with human DFF40 fused with GM-CSF for targeting acute myeloid leukemia (AML) cells. cDNA cloning of human DFF40 and GM-CSF was done and the chimeric gene GM-CSF-DFF40 was generated by overlap extension PCR. The fusion protein was expressed in E.coli, purified, refolded and characterized. In vitro cytotoxicity was evaluated on various AML cell lines. Treated cell lines were screened for various morphological and biochemical changes that are characteristic of apoptosis, by different assays like annexin V-FITC staining, TUNEL assay, JC-1 staining and immunocytochemistry of pro-apoptotic proteins and caspases. Cell cycle analysis of treated cells was done to quantify the percentage of apoptotic cells. The chimeric protein was found to be cytotoxic to AML cells in a dose and time dependent manner. Morphological changes such as formation of apoptotic bodies were revealed by microscopic examination of treated cells after staining. Immunocytochemical staining demonstrated biochemical changes such as changes in mitochondrial membrane potential, mitochondrial co-localization of Bax, cytochrome c release, presence of activated caspase-3 and DNA fragmentation. FACS analysis proved the presence of apoptotic cells following treatment. The chimeric protein GM-CSF-DFF40 was found to mediate targeted killing of AML cells by inducing apoptosis. Thus, this chimeric construct can act as a prospective candidate for targeted therapy of AML and other malignancies where GM-CSF receptor expression is upregulated.

Lower peripheral blood CD14+ monocyte frequency and higher CD34+ progenitor cell frequency are associated with HBV vaccine induced response in HIV infected individuals

We evaluated immunologic predictors of response to HBV vaccine administered in the presence or absence of GM-CSF in HIV infected individuals. We measured peripheral blood hematopoietic progenitor, monocyte and myeloid-derived suppressor cell (MDSC) frequencies, and expression of GMCSF receptor on monocytes and MDSCs, at baseline and 4weeks after immunization in relation to antibody response. We observed higher baseline progenitor and lower monocyte frequencies among week 16 antibody responders. Week 4 decline in MDSC frequency was associated with week 16 antibody response, while administration of GM-CSF was associated with preservation of these cells. No significant differences in GM-CSF receptor expression were observed in the presence vs. absence of GM-CSF. These findings are consistent with a positive role of progenitor cells and a potential negative role of monocytes in vaccine response. Additionally, GM-CSF augmented the preservation of peripheral blood MDSC, which may contribute to the lack of improved vaccine responses.