Granulocyte-macrophage Colony-stimulating Factor Treatment Research Articles

Synergism between cryoablation and GM-CSF: enhanced immune function of splenic dendritic cells in mice with glioma.

Glioma is the most common malignant primary brain tumor, and it has a poor prognosis. Studies have shown that cryoablation can activate antitumor immunoeffects by promoting the augmentation of dendritic cells (DCs). Granulocyte macrophage colony-stimulating factor (GM-CSF) has been shown to be useful for immunotherapy against glioma because it can stimulate DCs to present tumor antigen. Previous studies have shown that cryoablation and GM-CSF can exert antitumor effects. To test the hypothesis that combined therapy with cryoablation and GM-CSF for glioma could synergistically improve specific antiglioma immunity in mice, we tested the validity of this assumption in a murine subcutaneous GL261 glioma model. C57BL/6 mice with subcutaneous GL261 glioma were created and divided into four groups: no treatment, GM-CSF injection, cryoablation treatment, and GM-CSF and cryoablation combined treatment (n=20 in each group). Serial immune indicators were detected at sequential time points during treatment. Compared with the other groups, in the combined treatment group, DCs were more activated and their numbers were markedly upregulated, the secretion of interferon-γ from Th1 cells of mice spleen was increased, and the cytolytic activity of CD8 CTLs exerted a more significant cytotoxic effect on GL261 glioma cells (P<0.05 for all). Furthermore, these changes peaked on the 7th day after treatment, and then gradually reduced, until the 21st day; these changes were higher than those at pretreatment (P<0.05). It is concluded that combined therapy with argon-helium cryoablation and GM-CSF could synergistically enhance the activation of DCs and induce a robust tumor-specific immunologic response in glioma-bearing mice.

Adjuvant GM-CSF Improves Survival in High-risk Stage IIIC Melanoma

Stage III melanoma is associated with an increased risk of recurrence and death. Complete surgical resection remains the best chance for cure. Unfortunately, no adjuvant therapy has demonstrated a consistent improvement in melanoma-specific survival (MSS). We hypothesize that adjuvant granulocyte-macrophage colony-stimulating factor (GM-CSF) may improve clinical outcomes. Retrospective cohort study of 317 surgically resected stage III melanoma patients managed with observation or adjuvant GM-CSF at a single institution from 2001 to 2010. Of the 317 stage III patients, 165 (52%) were observed and 152 (48%) were treated with GM-CSF, with a median follow-up of 34 months. Patients treated with GM-CSF tended to be younger (P < 0.0001), had more advanced stage disease (P = 0.002), and were more likely to have had a recurrence before initiation of adjuvant therapy than the observation group (P < 0.0001). Adjuvant GM-CSF seemed to be associated with improved MSS, but this did not reach statistical significance (P = 0.08). Patients with stage IIIC melanoma derived a substantial benefit from adjuvant GM-CSF, with a 52% risk reduction in melanoma-specific death (hazard ratio 0.48; 95% confidence interval, 0.27-0.87; P = 0.02). Despite selecting patients with more advanced stage and a higher incidence of regional relapse, adjuvant GM-CSF was associated with an improved MSS but not disease-free survival in patients with stage IIIC disease. In patients not otherwise eligible for clinical trials, adjuvant GM-CSF treatment is a reasonable option for individuals with resected high-risk melanoma.

Homeobox Transcription Factor VentX Regulates Differentiation and Maturation of Human Dendritic Cells

Dendritic cells (DCs) are specialized antigen presentation cells that play critical roles in the initiation and regulation of immune responses. The molecular determinants of DC differentiation and maturation are target of extensive investigation. VentX is a human homeobox transcriptional factor that regulates proliferation and differentiation of hematopoietic cells. In the current study, we report that ablation of VentX expression in monocytes significantly impaired their differentiation into DCs. Conversely, overexpression of VentX in monocytic THP1 cells accelerated their differentiation toward DCs. We showed that VentX regulates DC differentiation, in part, through modulating IL6 expression. Clinically, we found that VentX expression was elevated in intestinal lamina propria DCs (LPDCs) of inflamed mucosa from inflammatory bowel disease patients. Knockdown experiments suggested that VentX is essential for the maturation of LPDCs. In addition, corticosteroid treatment markedly decreased VentX expression in LPDCs and enforced expression of VentX counteracted the effects of corticosteroid on DCs maturation. Our data suggest that VentX is a critical transcriptional regulator of DC differentiation and maturation, and a potential target of immune regulation and therapy.

Granulocyte/Macrophage Colony-Stimulating Factor Influences Angiogenesis by Regulating the Coordinated Expression of VEGF and the Ang/Tie System

Granulocyte/macrophage colony-stimulating factor (GM-CSF) can accelerate wound healing by promoting angiogenesis. The biological effects of GM-CSF in angiogenesis and the corresponding underlying molecular mechanisms, including in the early stages of primitive endothelial tubule formation and the later stages of new vessel maturation, have only been partially clarified. This study aimed to investigate the effects of GM-CSF on angiogenesis and its regulatory mechanisms. Employing a self-controlled model (Sprague-Dawley rats with deep partial-thickness burn wounds), we determined that GM-CSF can increase VEGF expression and decrease the expression ratio of Ang-1/Ang-2 and the phosphorylation of Tie-2 in the early stages of the wound healing process, which promotes the degradation of the basement membrane and the proliferation of endothelial cells. At later stages of wound healing, GM-CSF can increase the expression ratio of Ang-1/Ang-2 and the phosphorylation of Tie-2 and maintain a high VEGF expression level. Consequently, pericyte coverages were higher, and the basement membrane became more integrated in new blood vessels, which enhanced the barrier function of blood vessels. In summary, we report here that increased angiogenesis is associated with GM-CSF treatment, and we indicate that VEGF and the Ang/Tie system may act as angiogenic mediators of the healing effect of GM-CSF on burn wounds.

RUNX1-ETO Expressing Hematopoietic Stem/Progenitor Cells (HSPCs) Display a Hypersensitivity To GM-CSF That Reduces Leukemogenic Potential

RUNX1-ETO (also known as AML1-ETO and AML1-MTG8) is a fusion gene generated from t(8;21), which is a common chromosome translocation in acute myeloid leukemia (AML). It has been shown that t(8;21) requires additional aberrations to induce leukemia. Interestingly, 32-59% of t(8;21) patients also display loss of a sex chromosome (LOS) in their leukemia cells. Therefore, loss of the genes located on the sex chromosomes, especially in the pseudoautosomal regions (PARs) that are shared between the X and Y chromosomes, may contribute to RUNX1-ETO leukemia development. One gene of interest in the PARs is CSF2RA, which encodes the alpha subunit of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor. When the GM-CSF receptor is bound to its ligand, downstream signaling events promote various functional outcomes including proliferation, differentiation, self-renewal, and survival of myeloid cells. Thus, GM-CSF signaling has the potential to regulate both normal and malignant hematopoiesis. We previously reported that mice expressing RUNX1-ETO in GM-CSF deficient hematopoietic cells displayed higher incidence of leukemia (Matsuura S et al. 2012 Blood 119:3155). This result suggests that GM-CSF signaling is inhibitory to RUNX1-ETO dependent leukemogenesis. Furthermore, GM-CSF treatment reduces the self-renewal potential of RUNX1-ETO expressing cells and promotes myeloid differentiation in replating assays. We therefore hypothesize that the negative effect of GM-CSF on RUNX1-ETO induced leukemia development is due to the activation of selected GM-CSF downstream signaling pathway(s) that diminish self-renewal capacity and promote myeloid differentiation.To understand the molecular mechanism of the negative effect of GM-CSF on t(8;21) leukemogenesis, in the current report, we conducted a gene expression profiling assay to examine the effect of GM-CSF on RUNX1-ETO cells. MigR1 vector control or MigR1-RUNX1-ETO retrovirus transduced lineage negative/c-Kit positive (Lin-/c-Kit+) murine hematopoietic stem/progenitor cells (HSPCs) were cultured with or without GM-CSF for 24 hours. Then, Lin-/c-Kit+/GFP+ HSPCs were isolated for the profiling study. We observed little response to GM-CSF in control HSPCs, with only 4 genes being differentially expressed after a 2-fold cutoff. Conversely, 122 genes were differentially expressed in RUNX1-ETO cells treated with GM-CSF. These results clearly indicate that RUNX1-ETO specifically enhances GM-CSF responsiveness in HSPCs. Gene Set Enrichment Analysis (GSEA) of the differentially expressed genes in RUNX1-ETO cells reveals that this response resembles that of GM-CSF-induced myeloid differentiation. Furthermore, pathway analysis of these differentially expressed genes predicts MEK1/2 and ERK1/2 to be activated after GM-CSF treatment in RE cells. We previously reported that ERK1/2, downstream targets of MEK1/2, are hyper-phosphorylated after GM-CSF treatment of RUNX1-ETO cells, and MEK-ERK activation has been shown to regulate cell proliferation and myelopoiesis. Other GM-CSF induced genes are predicted targets of MYD88. MYD88 is upregulated during myeloid differentiation. Its in vivo knockout has been reported to result in an increase of hematopoietic stem cells (HSCs) and reduction of mature granulocytes. Most interestingly, a subset of genes upregulated in GM-CSF treated RUNX1-ETO cells are predicted to be activated by CEBPβ. CEBPβ can heterodimerize with CEBPα and is induced during myelopoiesis, critical for macrophage differentiation, capable of promoting granulopoiesis, and involved in regulating granulopoiesis in vivo.In conclusion, our data suggest that RUNX1-ETO expression results in hyper-responsiveness to GM-CSF. Such enhanced GM-CSF signaling activates the expression of a specific group of genes and results in the reduced self-renewal capacity and increased myeloid differentiation of HSPCs. These GM-CSF effects are likely involved in reducing the leukemogenic potential of RUNX1-ETO and may be considered for specific therapeutic interventions. Disclosures:No relevant conflicts of interest to declare.

Inhibitory effect of Korean Red Ginseng on melanocyte proliferation and its possible implication in GM-CSF mediated signaling

Korean Red Ginseng (KRG) has been reported to exert anticancer, anti-oxidant, and anti-inflammatory effects. However, there has been no report on the effect of KRG on skin pigmentation. In this study, we investigated the inhibitory effect of KRG on melanocyte proliferation. KRG extract (KRGE) at different concentrations had no effect on melanin synthesis in melan-A melanocytes. Saponin of KRG (SKRG) inhibited melanin content to 80% of the control at 100 ppm. Keratinocyte-derived factors induced by UV-irradiation were reported to stimulate melanogenesis, differentiation, proliferation, and dendrite formation. In this study, treatment of melan-A melanocytes with conditioned media from UV-irradiated SP-1 keratinocytes increased melanocyte proliferation. When UV-irradiated SP-1 keratinocytes were treated with KRGE or SKRG, the increase of melanocyte proliferation by the conditioned media was blocked. Granulocyte-macrophage colony-stimulating factor (GM-CSF) was produced and released from UV-irradiated keratinocytes. This factor has been reported to be involved in regulating the proliferation and differentiation of epidermal melanocytes. In this study, GM-CSF was significantly increased in SP-1 keratinocytes by UVB irradiation (30 mJ/cm2), and the proliferation of melan-A melanocytes increased significantly by GM-CSF treatment. In addition, the proliferative effect of keratinocyte-conditioned media on melan-A melanocytes was blocked by anti-GM-CSF treatment. KRGE or SKRG treatment decreased the expression of GM-CSF in SP-1 keratinocytes induced by UVB irradiation. These results demonstrate that UV irradiation induced GM-CSF expression in keratinocytes and KRGE or SKRG inhibited its expression. Therefore, KRG could be a good candidate for regulating UV-induced melanocyte proliferation.

Alveolar macrophages of GM-CSF knockout mice exhibit mixed M1 and M2 phenotypes

BackgroundActivin A is a pleiotrophic regulatory cytokine, the ablation of which is neonatal lethal. Healthy human alveolar macrophages (AMs) constitutively express activin A, but AMs of patients with pulmonary alveolar proteinosis (PAP) are deficient in activin A. PAP is an autoimmune lung disease characterized by neutralizing autoantibodies to Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF). Activin A can be stimulated, however, by GM-CSF treatment of AMs in vitro. To further explore pulmonary activin A regulation, we examined AMs in bronchoalveolar lavage (BAL) from wild-type C57BL/6 compared to GM-CSF knockout mice which exhibit a PAP-like histopathology. Both human PAP and mouse GM-CSF knockout AMs are deficient in the transcription factor, peroxisome proliferator activated receptor gamma (PPARγ).ResultsIn sharp contrast to human PAP, activin A mRNA was elevated in mouse GM-CSF knockout AMs, and activin A protein was increased in BAL fluid. Investigation of potential causative factors for activin A upregulation revealed intrinsic overexpression of IFNγ, a potent inducer of the M1 macrophage phenotype, in GM-CSF knockout BAL cells. IFNγ mRNA was not elevated in PAP BAL cells. In vitro studies confirmed that IFNγ stimulated activin A in wild-type AMs while antibody to IFNγ reduced activin A in GM-CSF knockout AMs. Both IFNγ and Activin A were also reduced in GM-CSF knockout mice in vivo after intratracheal instillation of lentivirus-PPARγ compared to control lentivirus vector. Examination of other M1 markers in GM-CSF knockout mice indicated intrinsic elevation of the IFNγ-regulated gene, inducible Nitrogen Oxide Synthetase (iNOS), CCL5, and interleukin (IL)-6 compared to wild-type. The M2 markers, IL-10 and CCL2 were also intrinsically elevated.ConclusionsData point to IFNγ as the primary upregulator of activin A in GM-CSF knockout mice which in addition, exhibit a unique mix of M1-M2 macrophage phenotypes.

Granulocyte Macrophage Colony-Stimulating Factor Shows Anti-apoptotic Activity via the PI3K–NF-κB–HIF-1α–Survivin Pathway in Mouse Neural Progenitor Cells

Granulocyte macrophage-colony stimulating factor (GM-CSF) is a hematopoietic cytokine that plays a crucial role in regulating the proliferation, differentiation, and survival of hematopoietic cells. Recent studies have shown that GM-CSF also has anti-apoptotic effects and regulates the expression of anti-apoptotic genes including Bcl-2 family proteins in neuronal cells in vitro and in vivo. However, the mechanism underlying the anti-apoptotic function of GM-CSF is not well understood. In the present work, we examined the role of phosphoinositide 3-kinase (PI3K)-AKT signal pathway in the anti-apoptotic activity of GM-CSF in mouse neural progenitor cells (NPCs). In terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, the anti-apoptotic effect of GM-CSF (apoptotic population of approximately 8.17 %) on staurosporine-induced apoptosis of NPCs (31.09 %) was significantly blocked by LY294002, an inhibitor of PI3K signal (24.04 %). We found that the PI3K-AKT signal pathway induced by GM-CSF treatment activated nuclear factor κB (NF-κB) and increased the expression of hypoxia-inducible factor 1α (HIF-1α) in normoxic conditions. Analyses using specific small interfering RNAs (siRNAs) showed that NF-κB was an upstream molecule of HIF-1α and activated its expression at the mRNA level. Further analyses using the siRNAs and chromatin immunoprecipitation (ChIP) showed that HIF-1α was responsible for the induced expression of survivin, a member of the inhibitor of apoptosis proteins (IAPs). Each of the specific siRNAs for NF-κB, HIF-1α, and survivin inhibited significantly the anti-apoptotic activity of GM-CSF on the staurosporine-induced apoptosis in NPCs in TUNEL assays. The results of this study showed the downstream signals and mechanism of PI3K/AKT-mediated anti-apoptotic activity of GM-CSF in NPCs, particularly revealing the role of the NF-κB-HIF-1α-survivin cascade.

Influence of granulocyte macrophagecolony stimulating factor on the expression of angiopoietin-1 in deep partial thickness burn wound in rats

Objective To investigate the influence of granulocyte macrophage colony stimulatingfactor （ GM-CSF） on the expression of angiopoietin-1 （Ang-1） in deep partial thickness bum wound in 1rats. Methods The models of deep partial thickness bum wounds on the back in Spregue-Dawley （ SD）rats were established. The paired wounds on the same rats were randomly divided into GM-CSF treatment（test） and control groups by self-control. Tissue samples were harvested from the wounds on the 1st,3rd,7th, 14th and 21st day respectively after bums. The mRNA expression of Ang-1 was detected by using re-al-time reverse transcriptase-polymerase chain reaction （ FQ-PCR） . And the protein expression of Ang-1was detected by immunohistochemical and western blot, the protein expression of CD31 by using immuno-histochemical staining,and the microvascular density was calculated. Results The mRNA expression levelof Ang-1 in the bum wounds was decreased slightly on the 1st and 3rd postbum days and that in test group（1. 44 ±0. 20,0. 30 ± 0. 17 ） was lower than that in control group （2. 36 ± 0. 18 , 1. 34 ± 0. 24） （P 〈0.05）. Then the Ang-1 mRNA expression was gradually increased on the 7 th and 14 th postbum days anddecreased slightly on the 21st postbum day, and the expression level in the test group at the above timepoints （4. 52 ±0. 21,8. 47 ±0. 14, 7. 52 ±0. 20） was higher than that in control group （2. 71 ±0. 12,3.69 ±0. 24,3. 07 ±0. 17）（p 〈0. 05）. The protein expression of Ang-1 was declined remarkably on the 1stand 3rd postburn days, which in test group （0. 491 ±0. 055, 0. 181 ±0. 025） was lower than that in control group （0. 735 ± O. 080, O. 436 ± 0. 071 ） （ P 〈 O. 05 ）. Then the Ang-1 protein expression was gradually in- creased and reached the peak on the 14th postburn day and the expression in the test group （0. 821 ±0. 060, 1. 257 ± 0. 225, 1. 126 ±0. 131 ） was higher than that in the control group （0. 701 ± 0. 078, 0. 825 ± 0. 072, 0. 733 ±0. 073 ） （P 〈 0. 05 ）. The microvascular number with positive CD31 expression was increased evi- dently from the 3rd postburn day in test group, and the counting of MVD was remarkably higher in the test group （9.5±1.8, 23.4±2.9, 38.4±3. 1,24.6±2.5） than in the control group （4.0±1.9, 10.5±2.0, 22. 9 ±2. 7, 15.1 ±2. 6） on the 3rd, 7th, 14th and 21st days respectively after burns （P 〈0. 05）. Conclu- sion GM-CSF inhibits the mRNA and protein expression of Ang-1 during the early stage of wound healing, which can be beneficial to the angiogenesis. And GM-CSF boosts Ang-1 expression during the middle and late stages of wound healing. This would be helpful to wound vascular maturation and wound healinz. Key words: Granulocyte macrophage colony stimulating factor; Angiopoietin; Burn; Woundhealing

GM‐CSF enhances tumor invasion by elevated MMP‐2, ‐9, and ‐26 expression

Granulocyte–macrophage colony-stimulating factor (GM-CSF) promotes tumor progression in different tumor models in an autocrine and paracrine manner. However, at the same time GM-CSF is used in cancer therapies to ameliorate neutropenia. We have previously shown in GM-CSF and G-CSF expressing or negative skin or head and neck squamous cell carcinoma that GM-CSF expression is associated with a highly angiogenic and invasive tumor phenotype. To determine the functional contribution of GM-CSF to tumor invasion, we stably transfected a GM-CSF negative colon adenocarcinoma cell line HT-29 with GM-CSF or treated the same cell line with exogenous GM-CSF. While GM-CSF overexpression and treatment reduced tumor cell proliferation and tumor growth in vitro and in vivo, respectively, it contributed to tumor progression. Together with an enhanced migratory capacity in vitro, we observed a striking increase in tumor cell invasion into the surrounding tissue concomitant with the induction of an activated tumor stroma in GM-CSF overexpressing or GM-CSF treated tumors. In a complex 3D in vitro model, enhanced GM-CSF expression was associated with a discontinued basement membrane deposition that might be mediated by the increased expression and activation of MMP-2, -9, and -26. Treatment with GM-CSF blocking antibodies reversed this effect. The increased presence and activity of these tumor cell derived proteases was confirmed in vivo. Here, expression of MMP-26 protein was predominantly located in pre- and early-invasive areas suggesting MMP-26 expression as an early event in promoting GM-CSF dependent tumor invasion.

Improvement in sensory function via granulocyte-macrophage colony-stimulating factor in rat spinal cord injury models

The aim in this study was to determine whether granulocyte-macrophage colony-stimulating factor (GM-CSF) leads to sensory improvement in rat spinal cord injury (SCI) models. Thirty male Sprague-Dawley rats were included in this study: 10 in the sham group (laminectomy alone without SCI), 10 in the SCI group (SCI treated with phosphate-buffered saline), and 10 in the GM-CSF treatment group (SCI treated with GM-CSF). A locomotor function test and pain sensitivity test were conducted weekly for 9 weeks after SCI or sham injury. Spinal tissue samples from all rats were immunohistochemically examined for the expression of calcitonin gene-related peptide (CGRP) and abnormal sprouting at Week 9 post-SCI. Granulocyte-macrophage colony-stimulating factor treatment improves functional recovery after SCI. In the tactile withdrawal threshold and frequency of the hindlimb paw, the GM-CSF group always responded with a statistically significant lower threshold than the SCI group 9 weeks after SCI (p < 0.05). The response of the forelimb and hindlimb paws to cold in the GM-CSF group always reflected a statistically significant lower threshold than in the SCI group 9 weeks after injury (p < 0.05). Decreased CGRP expression, observed by density and distribution area, was noted in the GM-CSF group (optical density 113.5 ± 20.4) compared with the SCI group (optical density 143.1 ± 18.7; p < 0.05). Treatment with GM-CSF results in functional recovery, improving tactile and cold sense recovery in a rat SCI model. Granulocyte-macrophage colony-stimulating factor also minimizes abnormal sprouting of sensory nerves after SCI.

Hypoxia Inducible Factors-Mediated Inhibition of Cancer by GM-CSF: A Mathematical Model

Under hypoxia, tumor cells, and tumor-associated macrophages produce VEGF (vascular endothelial growth factor), a signaling molecule that induces angiogenesis. The same macrophages, when treated with GM-CSF (granulocyte/macrophage colony-stimulating factor), produce sVEGFR-1 (soluble VEGF receptor-1), a soluble protein that binds with VEGF and inactivates its function. The production of VEGF by macrophages is regulated by HIF-1α (hypoxia inducible factor-1α), and the production of sVEGFR-1 is mediated by HIF-2α. Recent experiments measured the effect of inhibiting tumor growth by GM-CSF treatment in mice with HIF-1α-deficient or HIF-2α-deficient macrophages. In the present paper, we represent these experiments by a mathematical model based on a system of partial differential equations. We show that the model simulations agree with the above experiments. The model can then be used to suggest strategies for inhibiting tumor growth. For example, the model qualitatively predicts the extent to which GM-CSF treatment in combination with a small molecule inhibitor that stabilizes HIF-2α will reduce tumor volume and angiogenesis.

Chronic Alcohol Ingestion in Rats Decreases Krüppel‐Like Factor 4 Expression and Intracellular Zinc in the Lung

Chronic alcohol ingestion alters the dynamic balance between granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor beta1 (TGFβ1) signaling within the alveolar space and, in parallel, impairs alveolar macrophage and epithelial cell function by inhibiting expression of the zinc importer ZIP4 and decreasing zinc bioavailability in the alveolar compartment. As the transcription factor Krüppel-like factor 4 (KLF4 ) binds to ZIP4 , we hypothesized that alcohol exposure and consequent perturbations in GM-CSF and TGFβ1 signaling could decrease cellular KLF4 expression and/or binding as a mechanism by which it inhibits ZIP4 expression and decreases cellular zinc levels. Alcohol exposure in vitro or chronic ingestion in vivo decreased KLF4 expression in alveolar macrophages and epithelial cells. Treatment with GM-CSF or TGFβ1 showed an enhancing or dampening effect on KLF4 expression and binding, respectively. Further, treatment of a rat alveolar macrophage cell line with alcohol in vitro for 4weeks decreased the expression of the zinc transporters ZIP4 and ZNT1, and of the zinc storage protein metallothionein 1. In parallel, treating these macrophages with KLF4 siRNA decreased ZIP4 expression and decreased cellular zinc and phagocytic capacity to levels equivalent to those following alcohol exposure. In epithelial monolayers, transepithelial electrical resistance (TER) was significantly decreased by alcohol ingestion as compared with control diets, and it was restored by in vitro GM-CSF treatment. In contrast, in vitro TGFβ1 treatment of the epithelial monolayers from control-fed rats significantly decreased TER as compared with untreated control monolayers. Taken together, these results suggest that within the alveolar space, chronic alcohol exposure decreases KLF4 and ZIP4 expression and consequently decreases zinc transport into cells, which, in turn, impairs their function. Furthermore, the dynamic decrease in the relative influence of GM-CSF versus TGFβ1 could mediate the zinc deficiency and consequent cellular dysfunction that characterize the "alcoholic lung" phenotype.

Lactoferrin Regulates an Axis Involving CD11b and CD49d Integrins and the Chemokines MIP-1α and MCP-1 in GM-CSF-Treated Human Primary Eosinophils

Eosinophils are multifunctional immune cells that contribute to innate and adaptive immune/repair responses. Lactoferrin (LF) is an iron-binding protein indicated to alter cell adhesion and immune function by receptor-mediated interactions or by participating in redox mechanisms. The eosinophil adhesion molecules, αMβ2 and α4β1, are differentially expressed following exposure to the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) and various redox agents. We hypothesized that LF can alter the function and production of proteins involved in adhesion/migration. Utilizing eosinophil peroxidase activity or fluorescent labeling adhesion assays, LF reduced GM-CSF-induced eosinophil adhesion in the presence of fibronectin or vascular adhesion molecule-1 compared with GM-CSF treatment alone. Flow cytometric analysis of eosinophil αM (CD11b) and α4 (CD49d) integrins revealed that cotreatments (24 h) with LF plus GM-CSF induced a significant increase in CD11b compared with control and GM-CSF treatments but a significant decrease in CD49d compared with control and GM-CSF treatments. These changes in CD11b and CD49d levels were significantly correlated with the increased production of chemokines (macrophage inflammatory Protein-1α, monocyte chemotactic protein-1) and an identified increase in S100A9 production. Thus, LF release at sites of inflammation may alter eosinophil recruitment/activation and possibly the progression of diseases such as cancer and asthma where significant eosinophil influx has been described.

Molecular Mechanism of Hepcidin-Mediated Ferroportin Internalization Requires Ferroportin Lysines, Not Tyrosines or JAK-STAT

Ferroportin is the primary means of cellular iron efflux and a key component of iron metabolism. Hepcidin regulates Fpn activity by inducing its internalization and degradation. The mechanism of internalization is reported to require JAK2 activation, phosphorylation of Fpn tyrosine residues 302 and 303, and initiation of transcription through STAT3 phosphorylation. These findings suggest Fpn may be a target for therapeutic intervention through JAK2 modulation. To evaluate the proposed mechanism, Fpn internalization was assessed using several techniques combined with reagents that specifically recognized cell-surface Fpn. Invitro results demonstrated that Hepc-induced Fpn internalization did not require JAK2 or phosphorylation of Fpn residues 302 and 303, nor did it induce JAK-STAT signaling. Invivo, inhibition of JAK2 had no effect on Hepc-induced hypoferremia. However, internalization was delayed by mutation of two Fpn lysine residues that may be targets of ubiquitination.

Molecular cloning and functional characterization of a mouse ccl6 analog gene in the rat

Suppression subtractive hybridization was used to analyze differential expression of genes in rat peritoneal macrophages after granulocyte macrophage colony-stimulating factor treatment. We identified and cloned the mouse C10 analog gene in the rat, and named it as ccl6. The full-length cDNA of rat ccl6 was 467 bp, which contains a single-open reading frame and encodes 116 amino acid residues. Compared with other C-C chemokines, the rat ccl6 gene had an unusual four-exon genome structure instead of the typical three exons, it had the highest homology with murine ccl6. The rat ccl6 gene was localized on chromosome 10, where most of the C-C chemokine superfamily members are located. The recombinant rat C-C chemokine ligand 6 (CCL6) protein was expressed by the pGEX4T-1 plasmid in Escherichia coli BL21. The purified recombinant protein had bioactivity similar to that of mouse CCL6, which is a chemoattractant for macrophages and lymphocytes, but not for neutrophils.

A randomized trial of recombinant human granulocyte-macrophage colony stimulating factor for patients with acute lung injury*

Despite recent advances in critical care and ventilator management, acute lung injury and acute respiratory distress syndrome continue to cause significant morbidity and mortality. Granulocyte-macrophage colony stimulating factor may be beneficial for patients with acute respiratory distress syndrome. To determine whether intravenous infusion of granulocyte-macrophage colony stimulating factor would improve clinical outcomes for patients with acute lung injury/acute respiratory distress syndrome. A randomized, double-blind, placebo-controlled clinical trial of human recombinant granulocyte-macrophage colony stimulating factor vs. placebo. The primary outcome was days alive and breathing without mechanical ventilatory support within the first 28 days after randomization. Secondary outcomes included mortality and organ failure-free days. Medical and surgical intensive care units at three academic medical centers. One hundred thirty individuals with acute lung injury of at least 3 days duration were enrolled, out of a planned cohort of 200 subjects. Patients were randomized to receive human recombinant granulocyte-macrophage colony stimulating factor (64 subjects, 250 μg/M) or placebo (66 subjects) by intravenous infusion daily for 14 days. Patients received mechanical ventilation using a lung-protective protocol. There was no difference in ventilator-free days between groups (10.7 ± 10.3 days placebo vs. 10.8 ± 10.5 days granulocyte-macrophage colony stimulating factor, p = .82). Differences in 28-day mortality (23% in placebo vs. 17% in patients receiving granulocyte-macrophage colony stimulating factor (p = .31) and organ failure-free days (12.8 ± 11.3 days placebo vs. 15.7 ± 11.9 days granulocyte-macrophage colony stimulating factor, p = .16) were not statistically significant. There were similar numbers of serious adverse events in each group. In a randomized phase II trial, granulocyte-macrophage colony stimulating factor treatment did not increase the number of ventilator-free days in patients with acute lung injury/acute respiratory distress syndrome. A larger trial would be required to determine whether treatment with granulocyte-macrophage colony stimulating factor might alter important clinical outcomes, such as mortality or multiorgan failure. (ClinicalTrials.gov number, NCT00201409 [ClinicalTrials.gov]).

A Case of Pulmonary Alveolar Proteinosis Improved with Inhaled Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF)

Pulmonary alveolar proteinosis (PAP) is a rare lung disease characterized by the accumulation of lipoproteinaceous material within the alveoli. Several studies have recently found that autoantibodies against granulocyte-macrophage colony-stimulating factor (GM-CSF) play a major role in the pathogenesis of idiopathic PAP. Consequently, inhaled or systemic injection of GM-CSF has been suggested as a promising treatment for PAP. A 54-year-old male visited our hospital for progressive dyspnea. Four years earlier, he was diagnosed with PAP based on a surgical lung biopsy in another institution. Whole-lung lavage was performed four times before he visited our hospital. We administered high-dose inhaled GM-CSF therapy for 12 weeks followed by 12 weeks of low-dose therapy. After the GM-CSF treatment, the patient’s symptoms, lung function, and radiological findings were improved significantly. (Korean J Med 2012;82:357-361)

GM-CSF provides autocrine protection for murine alveolar epithelial cells from oxidant-induced mitochondrial injury

Exposure of mice to hyperoxia induces alveolar epithelial cell (AEC) injury, acute lung injury and death. Overexpression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the lung protects against these effects, although the mechanisms are not yet clear. Hyperoxia induces cellular injury via effects on mitochondrial integrity, associated with induction of proapoptotic members of the Bcl-2 family. We hypothesized that GM-CSF protects AEC through effects on mitochondrial integrity. MLE-12 cells (a murine type II cell line) and primary murine type II AEC were subjected to oxidative stress by exposure to 80% oxygen and by exposure to H(2)O(2). Exposure to H(2)O(2) induced cytochrome c release and decreased mitochondrial reductase activity in MLE-12 cells. Incubation with GM-CSF significantly attenuated these effects. Protection induced by GM-CSF was associated with Akt activation. GM-CSF treatment also resulted in increased expression of the antiapoptotic Bcl-2 family member, Mcl-1. Primary murine AEC were significantly more tolerant of oxidative stress than MLE-12 cells. In contrast to MLE-12 cells, primary AEC expressed significant GM-CSF at baseline and demonstrated constitutive activation of Akt and increased baseline expression of Mcl-1. Treatment with exogenous GM-CSF further increased Akt activation and Mcl-1 expression in primary AEC. Conversely, suppression of AEC GM-CSF expression by use of GM-CSF-specific small interfering RNA resulted in decreased tolerance of oxidative stress, Furthermore, silencing of Mcl-1 prevented GM-CSF-induced protection. We conclude that GM-CSF protects alveolar epithelial cells against oxidative stress-induced mitochondrial injury via the Akt pathway and its downstream components, including Mcl-1. Epithelial cell-derived GM-CSF may contribute to intrinsic defense mechanisms limiting lung injury.

In vivo monitoring of pH, redox status, and glutathione using L‐band EPR for assessment of therapeutic effectiveness in solid tumors

Approach for in vivo real-time assessment of tumor tissue extracellular pH (pH(e)), redox, and intracellular glutathione based on L-band EPR spectroscopy using dual function pH and redox nitroxide probe and disulfide nitroxide biradical, is described. These parameters were monitored in PyMT mice bearing breast cancer tumors during treatment with granulocyte macrophage colony-stimulating factor. It was observed that tumor pH(e) is about 0.4 pH units lower than that in normal mammary gland tissue. Treatment with granulocyte macrophage colony-stimulating factor decreased the value of pH(e) by 0.3 units compared with PBS control treatment. Tumor tissue reducing capacity and intracellular glutathione were elevated compared with normal mammary gland tissue. Granulocyte macrophage colony-stimulating factor treatment resulted in a decrease of the tumor tissue reducing capacity and intracellular glutathione content. In addition to spectroscopic studies, pH(e) mapping was performed using recently proposed variable frequency proton-electron double-resonance imaging. The pH mapping superimposed with MRI image supports probe localization in mammary gland/tumor tissue, shows high heterogeneity of tumor tissue pH(e) and a difference of about 0.4 pH units between average pH(e) values in tumor and normal mammary gland. In summary, the developed multifunctional approach allows for in vivo, noninvasive pH(e), extracellular redox, and intracellular glutathione content monitoring during investigation of various therapeutic strategies for solid tumors.