Action Of Granulocyte Colony-stimulating Factor Research Articles

Mode of action of granulocyte-colony stimulating factor (G-CSF) as a novel therapy for stroke in a mouse model

BackgroundThe FDA approved drug granulocyte-colony stimulating factor (G-CSF) displays anti-apoptotic and immunomodulatory properties with neurogenesis and angiogenic functions. It is known to demonstrate neuroprotective mechanisms against ischemic global stroke. Autophagy is a method for the degradation of intracellular components and in particular, unrestrained autophagy may lead to uncontrolled digestion of affected neurons as well as neuronal death in cerebral ischemia. Mitochondrial dynamics is vital for the regulation of cell survival and death after cerebral ischemia and an early upstream event in neuronal death is mitochondrial fission. We examined the pro-survival mechanisms of G-CSF against apoptosis resulting from autophagy, mitochondrial stress and endoplasmic reticulum (ER) stress.MethodsMale Swiss Webster mice (20 weeks of age) were subjected to bilateral common carotid artery occlusion (BCAO) for 30 min. After occlusion, mice were injected with G-CSF (50 μg/kg) subcutaneously for 4 days. Behavioral analysis was carried out using the corner test and locomotor activity test before animals were sacrificed on day 4 or day 7. Key proteins in ER stress, autophagy and mitochondrial stress induced apoptosis were analyzed by immunoblotting.ResultsG-CSF improved neurological deficits and improved behavioral performance on corner and locomotor test. G-CSF binds to G-CSF receptors and its activation leads to upregulation of Akt phosphorylation (P-Akt) which in turn decreases levels of the ER stress sensor, GRP 78 and expression of proteins involved in ER stress apoptosis pathway; ATF6, ATF4, eIF2α, XBP1, Caspase 12 and CHOP. G-CSF treatment significantly decreased Beclin-1, an autophagy marker, and decreased mitochondrial stress biomarkers DRP1 and P53. G-CSF also up-regulated the mitochondrial fusion protein, OPA1 and anti-apoptotic protein Bcl-2 while down-regulating the pro-apoptotic proteins Bax, Bak and PUMA.ConclusionsG-CSF is an endogenous ligand in the CNS that has a dual activity that is beneficial both in reducing acute neuronal degeneration and adding to long-term plasticity after cerebral ischemia. G-CSF treatment exerts neuroprotective effects on damaged neurons through the suppression of the ER stress and mitochondrial stress and maintains cellular homeostasis by decreasing pro-apoptotic proteins and increasing of anti-apoptotic proteins.

Granulocyte-colony stimulating factor controls neural and behavioral plasticity in response to cocaine

Cocaine addiction is characterized by dysfunction in reward-related brain circuits, leading to maladaptive motivation to seek and take the drug. There are currently no clinically available pharmacotherapies to treat cocaine addiction. Through a broad screen of innate immune mediators, we identify granulocyte-colony stimulating factor (G-CSF) as a potent mediator of cocaine-induced adaptations. Here we report that G-CSF potentiates cocaine-induced increases in neural activity in the nucleus accumbens (NAc) and prefrontal cortex. In addition, G-CSF injections potentiate cocaine place preference and enhance motivation to self-administer cocaine, while not affecting responses to natural rewards. Infusion of G-CSF neutralizing antibody into NAc blocks the ability of G-CSF to modulate cocaine’s behavioral effects, providing a direct link between central G-CSF action in NAc and cocaine reward. These results demonstrate that manipulating G-CSF is sufficient to alter the motivation for cocaine, but not natural rewards, providing a pharmacotherapeutic avenue to manipulate addictive behaviors without abuse potential.

Granulocyte Colony—Stimulating Factor Multiplies Normal Blood ROS Generation at Less than 1 µg/l

Background: The neutrophils (PMN) are our main blood cells to combat fungi, bacteria, and fibrin. For normal function, an activated PMN generates a certain concentration of reactive oxygen species (ROS). If the generated blood ROS concentration is too low, then fungi, bacteria or fibrin might threaten the life of the patient, and it could be of great medical interest to stimulate PMN by physiologic drugs. Granulocyte-Colony Stimulating Factor (G-CSF) is a cell hormone that increases the cell number of PMN and that stimulates the individual PMN. The blood ROS generation assay (BRGA) is an innovative physiologic test to monitor the ROS generation of PMN in blood. Here the ROS generating action of G-CSF on normal PMN is quantified. Material and Methods: 40 μl 0 - 10.3 ng/ml (final conc.) G-CSF (in 5% human albumin) in black Brand? 781608 high quality polystyrene F-microwells was incubated in triplicate with 125 μl Hanks’ balanced salt solution (HBSS; modified without phenol red) and 10 μl normal citrated blood. Immediately (BRGA) or after 60 min (BRGA-60-) 10 μl 5 mM luminol sodium salt in 0.9% NaCl and 10 μl 0 or 36 μg/ml zymosan A in 0.9% NaCl was added. The photons were counted within 0 - 318 min (37°C) in a photons-multiplying microtiter plate luminometer. At about 0.5 t-maxn (0.5 fold the time to normal maximum) the approx. SC200 of G-CSF was determined. Results and Discussion: The approx. SC200 of G-CSF on normal blood ROS generation was 0.2 μg/l (=20 IU/ml). In clinical situations where an increased blood ROS generation is pharmacologically required, few micrograms of G-CSF could be a sufficient dosage for an adult patient. The BRGA helps to find out the correct stimulating G-CSF dosage for each individual. An enhanced PMN function could favor a better clinical outcome in situations of wanted increase of the innate immunology or in cellular fibrinolysis. G-CSF plasma concentrations of 0.1 - 1 μg/l might favor singlet oxygen generation without immunosuppression or cell fragment-induced thrombin generation.

Effects of an Inhibitor of Monocyte Recruitment on Recovery from Traumatic Brain Injury in Mice Treated with Granulocyte Colony-Stimulating Factor.

Administration of the hematopoietic growth factor granulocyte-colony stimulating Factor (G-CSF) has been reported to enhance recovery from controlled cortical impact (CCI) in rodent models. G-CSF exerts actions in both the periphery (stimulation of hematopoiesis) and in the brain, where it serves as a neurotrophic factor, promoting neuronal survival and stimulating neural stem/progenitor cell proliferation in the hippocampus. In order to distinguish the direct CNS actions of G-CSF from its peripheral actions, experiments were designed to block the recruitment of peripheral monocytes to the site of the lesion produced by CCI. The selective C-C motif receptor 2 (CCR2) antagonist (RS504303) was co-administered with G-CSF for three days after CCI in a chimeric mouse previously transplanted with GFP-expressing (GFP+) blood stem-progenitor cells. Results: The drug significantly impaired infiltration of GFP+ bone marrow-derived cells to the frontal cortex and striatum without impeding recovery performance and hippocampal neurogenesis in the behavioral test, the Radial Arm Water Maze (RAWM). Administration of the CCR2 antagonist alone, without G-CSF, was effective in promoting recovery in RAWM. These results support the hypothesis that the direct action of G-CSF on neural cells, independent of its hematopoietic effects, is primarily responsible for enhanced recovery from CCI. In addition, this study confirms the importance of CCR2 and its ligand, monocyte chemotactic protein-1 (MCP-1), in mediating the inflammatory response following CCI.

Local injection of granulocyte-colony stimulating factor accelerates wound healing in a rat excisional wound model.

A systemic treatment of granulocyte-colony stimulating factor (G-CSF) is known to improve healings of damaged tissues. However, recent studies suggested local actions of G-CSF on the healing processes of damaged tissues. We investigated the treatment effect of locally injected G-CSF and compared to that of systemically injected G-CSF in a rat model. A wound was created on the rat dorsum and treated either by local injection or by systemic injection of G-CSF. Wound healing rate, deposition of collagen, and gene expression were evaluated. G-CSF receptor (G-CSFR) protein was detected by Western blotting. The wound healing rate in the local injection group was significantly higher than that in the systemic injection group at days 9 and 15; it was also significantly higher than that in the control group at days 3, 9, and 15. The expression of G-CSFR protein in wound tissues was higher than in normal skin tissues. The local injection of G-CSF is more effective than systemic injection of G-CSF in promoting wound healing, which may implicate the local action of G-CSF treatment in wound healing processes.

Granulocyte colony-stimulating factor attenuates spinal cord injury-induced mechanical allodynia in adult rats

Spinal cord injury (SCI) can cause neuropathic pain (NeP), often reducing a patient's quality of life. We recently reported that granulocyte colony-stimulating factor (G-CSF) could attenuate NeP in several SCI patients. However, the mechanism of action underlying G-CSF-mediated attenuation of SCI-NeP remains to be elucidated.The purpose of the present study was to elucidate the therapeutic effect and mechanism of action of granulocyte colony-stimulating factor for SCI-induced NeP.T9 level contusive SCI was introduced to adult male Sprague Dawley rats. Three weeks after injury, rats received intraperitoneal recombinant human G-CSF (15.0μg/kg) for 5days. Mechanical allodynia was assessed using von Frey filaments. Immunohistochemistry and western blot analysis were performed in spinal cord lumbar enlargement samples.Testing with von Frey filaments showed significant increase in the paw withdrawal threshold in the G-CSF group compared with the vehicle group 4weeks, 5weeks, 6weeks and 7weeks after injury. Immunohistochemistry for CD11b (clone OX-42) revealed that the number of OX-42-positive activated microglia was significantly smaller in the G-CSF group than that in the vehicle rats. Western blot analysis indicated that phosphorylated-p38 mitogen-activated protein kinase (p38MAPK) and interleukin-1β expression in spinal cord lumbar enlargement were attenuated in the G-CSF-treated rats compared with that in the vehicle-treated rats.The present results demonstrate a therapeutic effect of G-CSF treatment for SCI-induced NeP, possibly through the inhibition of microglial activation and the suppression of p38MAPK phosphorylation and the upregulation of interleukin-1β.

RARγ is a negative regulator of osteoclastogenesis

Vitamin A is known to influence post-natal bone content, with excess intake being associated with reduced bone mineral density and increased fracture risk. Despite this, the roles retinoids play in regulating osteoclastogenesis, particularly in vivo, remain unresolved. This study therefore aimed to determine the effect of loss of retinoic acid receptors (RAR)α or RARγ on bone mass (analyzed by histomorphometry and dual-energy X-ray absorptiometry) and osteoclastogenesis in mice in vivo. RARγ null mice had significantly less trabecular bone at 8 weeks of age compared to wildtype littermates. In contrast, no change in trabecular bone mass was detected in RARα null mice at this age. Further histomorphometric analysis revealed a significantly greater osteoclast surface in bones from 8-week-old RARγ null male mice. This in vivo effect was cell lineage autonomous, and was associated with increased osteoclastogenesis in vitro from hematopoietic cells obtained from 8-week-old RARγ null male mice. The use of highly selective agonists in RANKL-induced osteoclast differentiation of wild type mouse whole bone marrow cells and RAW264.7 cells in vitro showed a stronger inhibitory effect of RARγ than RARα agonists, suggesting that RARγ is a more potent inhibitor of osteoclastogenesis. Furthermore, NFAT activation was also more strongly inhibited by RARγ than RARα agonists. While RARα and RARγ antagonists did not significantly affect osteoclast numbers in vitro, larger osteoclasts were observed in cultures stimulated with the antagonists, suggesting increased osteoclast fusion. Further investigation into the effect of retinoids in vivo revealed that oral administration of 5mg/kg/day ATRA for 10 days protected against bone loss induced by granulocyte colony-stimulating factor (G-CSF) by inhibiting the pro-osteoclastogenic action of G-CSF. Collectively, our data indicates a physiological role for RARγ as a negative regulator of osteoclastogenesis in vivo and in vitro, and reveals distinct influences of RARα and RARγ in bone structure regulation.

Analytical sequence to study G-CSF effect on the transcriptome of isolated spinal motoneurons from SOD1 G93A mice, an animal model for amyotrophic lateral sclerosis.

Granulocyte-colony stimulating factor (G-CSF) has been recently identified as a neurotrophic factor able to preserve motor functions, rescue motor units and extent survival in an animal model of amyotrophic lateral sclerosis, the SOD1 G93A mice. To gain insight into the mode of action of G-CSF, we have recently performed gene expression profiling on isolated lumbar motoneurons from SOD1G93A mice, and shown that G-CSF re-adjusted gene expression in motoneurons of symptomatic SOD1G93A mice and modulates genes related to neuromuscular function (Henriques et al., 2015). Here, we provide quality controls for the microarray experiment (GO accession number GSE60856) and describe the experimental strategy.

G-CSF treatment can attenuate dexamethasone-induced reduction in C2C12 myotube protein synthesis

Granulocyte-colony stimulating factor (G-CSF) has been demonstrated to enhance skeletal muscle recovery following injury and increases muscle function in the context of neuromuscular disease in rodent models. However, understanding of the underlying mechanisms used by G-CSF to mediate these functions remains poor. G-CSF acts on responsive cells through binding to a specific membrane spanning receptor, G-CSFR. Recently identified, the G-CSFR is expressed in myoblasts, myotubes and mature skeletal muscle tissue. Therefore, elucidating the actions of G-CSF in skeletal muscle represents an important prerequisite to consider G-CSF as a therapeutic agent to treat skeletal muscle. Here we show for the first time that treatment with moderate doses (4 and 40ng/ml) of G-CSF attenuates the effects of dexamethasone in reducing protein synthesis in C2C12 myotubes. However, a higher dose (100ng/ml) of G-CSF exacerbates the dexamethasone-induced reduction in protein synthesis. In contrast, G-CSF had no effect on basal or dexamethasone-induced protein degradation, nor did G-CSF influence the phosphorylation of Akt, STAT3, Erk1/2, Src, Lyn and Erk5 in C2C12 myotubes. In conclusion, physiologically relevant doses of G-CSF may attenuate reduced skeletal muscle protein synthesis during catabolic conditions, thereby improving recovery.

Gene expression changes in spinal motoneurons of the SOD1G93A transgenic model for ALS after treatment with G-CSF

Background: Amyotrophic lateral sclerosis (ALS) is an incurable fatal motoneuron disease with a lifetime risk of approximately 1:400. It is characterized by progressive weakness, muscle wasting, and death ensuing 3–5 years after diagnosis. Granulocyte-colony stimulating factor (G-CSF) is a drug candidate for ALS, with evidence for efficacy from animal studies and interesting data from pilot clinical trials. To gain insight into the disease mechanisms and mode of action of G-CSF, we performed gene expression profiling on isolated lumbar motoneurons from SOD1G93A mice, the most frequently studied animal model for ALS, with and without G-CSF treatment.Results: Motoneurons from SOD1G93A mice present a distinct gene expression profile in comparison to controls already at an early disease stage (11 weeks of age), when treatment was initiated. The degree of deregulation increases at a time where motor symptoms are obvious (15 weeks of age). Upon G-CSF treatment, transcriptomic deregulations of SOD1G93A motoneurons were notably restored. Discriminant analysis revealed that SOD1 mice treated with G-CSF has a transcriptom close to presymptomatic SOD1 mice or wild type mice. Some interesting genes modulated by G-CSF treatment relate to neuromuscular function such as CCR4-NOT or Prss12.Conclusions: Our data suggest that G-CSF is able to re-adjust gene expression in symptomatic SOD1G93A motoneurons. This provides further arguments for G-CSF as a promising drug candidate for ALS.

G-CSF does not influence C2C12 myogenesis despite receptor expression in healthy and dystrophic skeletal muscle.

Granulocyte-colony stimulating factor (G-CSF) increases recovery of rodent skeletal muscles after injury, and increases muscle function in rodent models of neuromuscular disease. However, the mechanisms by which G-CSF mediates these effects are poorly understood. G-CSF acts by binding to the membrane spanning G-CSFR and activating multiple intracellular signaling pathways. Expression of the G-CSFR within the haematopoietic system is well known, but more recently it has been demonstrated to be expressed in other tissues. However, comprehensive characterization of G-CSFR expression in healthy and diseased skeletal muscle, imperative before implementing G-CSF as a therapeutic agent for skeletal muscle conditions, has been lacking. Here we show that the G-CSFR is expressed in proliferating C2C12 myoblasts, differentiated C2C12 myotubes, human primary skeletal muscle cell cultures and in mouse and human skeletal muscle. In mdx mice, a model of human Duchenne muscular dystrophy (DMD), G-CSF mRNA and protein was down-regulated in limb and diaphragm muscle, but circulating G-CSF ligand levels were elevated. G-CSFR mRNA in the muscles of mdx mice was up-regulated however steady-state levels of the protein were down-regulated. We show that G-CSF does not influence C2C12 myoblast proliferation, differentiation or phosphorylation of Akt, STAT3, and Erk1/2. Media change alone was sufficient to elicit increases in Akt, STAT3, and Erk1/2 phosphorylation in C2C12 muscle cells and suggest previous observations showing a G-CSF increase in phosphoprotein signaling be viewed with caution. These results suggest that the actions of G-CSF may require the interaction with other cytokines and growth factors in vivo, however these data provides preliminary evidence supporting the investigation of G-CSF for the management of muscular dystrophy.

Granulocyte-colony stimulating factor reduces cardiomyocyte apoptosis and ameliorates diastolic dysfunction in Otsuka Long-Evans Tokushima Fatty rats.

In recent studies, granulocyte-colony stimulating factor (G-CSF) was shown to improve cardiac function in myocardial infarction and non-ischemic cardiomyopathies. The mechanisms of these beneficial effects of G-CSF in diabetic cardiomyopathy are not yet fully understood. Therefore, we investigated the mechanisms of action of G-CSF on diabetic cardiomyopathy in a rat model of type 2 diabetes. Seventeen-week-old OLETF (Otsuka Long Evans Tokushima Fatty) diabetic rats and LETO (Long Evans Tokushima Otuska) rats were randomized to treatment with 5days of G-CSF (100μg/kg/day) or with saline. Cardiac function was evaluated by serial echocardiography performed before and 4weeks after treatment. We measured expression of the G-CSF receptor (GCSFR) and Bcl-2, as well as the extent of apoptosis in the myocardium. G-CSF treatment significantly improved cardiac diastolic function in the serial echocardiography assessments. Expression of G-CSFR was down-regulated in the diabetic myocardium (0.03 ± 0.12% vs. 1 ± 0.15%, p < 0.05), and its expression was stimulated by G-CSF treatment (0.03 ± 0.12% vs. 0.42 ± 0.06%, p < 0.05). In addition, G-CSF treatment increased the expression of Bcl-2 in the diabetic myocardium (0.69 ± 0.06% vs. 0.26 ± 0.11%, p < 0.05), consistent with the reduced cardiomyocyte apoptosis (9.38 ± 0.67% vs. 17.28 ± 2.16%, p < 0.05). Our results suggest that G-CSF might have a cardioprotective effect in diabetic cardiomyopathy through up-regulation of G-CSFR, attenuation of apoptosis by up-regulation of Bcl-2 expression, and glucose-lowering effect. Our findings support the therapeutic potential of G-CSF in diabetic cardiomyopathy.

Efficacy of granulocyte-colony stimulating factor treatment in a rat model of anterior ischemic optic neuropathy.

Non-arteritic anterior ischemic optic neuropathy (NA-AION) is the most common cause of acute ischemic damage to the optic nerve (ON), and the leading cause of seriously impaired vision in people over 55 years of age. It demonstrated that subcutaneous administration of Granulocyte colony-stimulating factor (G-CSF) reduces RGC death in an ON crush model in rats, and that the neuroprotective effects may involve both anti-apoptotic and anti-inflammatory processes. Our recent work shows that the protective actions of G-CSF in rAION models may involve both anti-apoptotic and anti-inflammatory processes. However, the exact rescuing mechanisms involved in the administration of G-CSF in rAION models need further investigation. In addition, further studies on the administration of G-CSF at different time intervals after the induction of rAION may be able to illustrate whether treatment given at a later time is still neuroprotective. Further, it is unknown whether treatment using G-CSF combined with other drugs will result in a synergistic effect in a rAION model. Inflammation induced by ischemia plays an essential role on the ON head in NA-AION, which can result in disc edema and compartment changes. Therefore, it is reasonable that adding an anti-inflammatory drug may enhance the therapeutic effects of G-CSF. An ongoing goal is to evaluate the novel sites of action of both G-CSF and other anti-inflammatory drugs, and to identify the functionally protective pathways to enhance RGC survival. These investigations may open up new therapeutic avenues for the treatment of ischemic optic neuropathy.

Direct Actions of Granulocyte-Colony Stimulating Factor on Human Neuronal and Monocytic Cell Lines

Introduction: Granulocyte colony stimulating factor (G-CSF) administration produces beneficial effects in rodent models of stroke, trauma and neurodegenerative diseases by acting on both bone marrow-derived and neuronal cells. The aim of the study was to elucidate cellular mechanism(s) of G-CSF action by direct application to neuronal and monocytic cell lines. Method: Cell culture models of monocytes (THP-1) and neurons (SH-SY5Y) cells were incubated with G-CSF. The following parameters were measured: G-CSF receptor binding kinetics; DNA synthesis; signal transduction, in particular expression of alternatively spliced protein kinase C (PKCδVIII) and the anti-apoptotic protein Bcl-2; changes in adhesiveness and migratory properties induced by G-CSF in the monocytic cells. Results: G-CSF receptor binding kinetics in the two lines differed, with Kd in the neuronal line being significantly higher than that of the monocytic cells. Despite higher affinity of G-CSF for receptors on the monocytic cells, G-CSF treatment increased Bcl-2 expression in the neuronal line at lower concentrations than that required in the monocytic cell line. G-CSF did not increase either cellular adhesiveness or migration through a semi-permeable membrane, whereas monocyte chemotactic protein (MCP-1) significantly improved migration. Conclusions: The cellular and molecular responses to G-CSF treatment of monocytic cells suggest that neither changes in adhesiveness nor migratory capacity are responsible for the beneficial effects of G-CSF administration in models of neurologic diseases. G-CSF induction of anti-apoptotic signaling in neurons is an important component of its neuroprotective effects in models of brain injury.

Pharmacokinetic and -dynamic modelling of G-CSF derivatives in humans

BackgroundThe human granulocyte colony-stimulating factor (G-CSF) is routinely applied to support recovery of granulopoiesis during the course of cytotoxic chemotherapies. However, optimal use of the drug is largely unknown. We showed in the past that a biomathematical compartment model of human granulopoiesis can be used to make clinically relevant predictions regarding new, yet untested chemotherapy regimen. In the present paper, we aim to extend this model by a detailed pharmacokinetic and -dynamic modelling of two commonly used G-CSF derivatives Filgrastim and Pegfilgrastim.ResultsModel equations are based on our physiological understanding of the drugs which are delayed absorption of G-CSF when applied to the subcutaneous tissue, dose-dependent bioavailability, unspecific first order elimination, specific elimination in dependence on granulocyte counts and reversible protein binding. Pharmacokinetic differences between Filgrastim and Pegfilgrastim were modelled as different parameter sets. Our former cell-kinetic model of granulopoiesis was essentially preserved, except for a few additional assumptions and simplifications. We assumed a delayed action of G-CSF on the bone marrow, a delayed action of chemotherapy and differences between Filgrastim and Pegfilgrastim with respect to stimulation potency of the bone marrow. Additionally, we incorporated a model of combined action of Pegfilgrastim and Filgrastim or endogenous G-CSF which interact via concurrent receptor binding. Unknown pharmacokinetic or cell-kinetic parameters were determined by fitting the predictions of the model to available datasets of G-CSF applications, chemotherapy applications or combinations of it. Data were either extracted from the literature or were received from cooperating clinical study groups. Model predictions fitted well to both, datasets used for parameter estimation and validation scenarios as well. A unique set of parameters was identified which is valid for all scenarios considered. Differences in pharmacokinetic parameter estimates between Filgrastim and Pegfilgrastim were biologically plausible throughout.ConclusionWe conclude that we established a comprehensive biomathematical model to explain the dynamics of granulopoiesis under chemotherapy and applications of two different G-CSF derivatives. We aim to apply the model to a large variety of chemotherapy regimen in the future in order to optimize corresponding G-CSF schedules or to individualize G-CSF treatment according to the granulotoxic risk of a patient.

Suppression of dendritic cell functions contributes to the anti-inflammatory action of granulocyte-colony stimulating factor in experimental stroke

Cerebral ischemia provokes an inflammatory cascade, which is assumed to secondarily worsen ischemic tissue damage. Linking adaptive and innate immunity dendritic cells (DCs) are key regulators of the immune system. The hematopoietic factor G-CSF is able to modulate DC-mediated immune processes. Although G-CSF is under investigation for the treatment of stroke, only limited information exists about its effects on stroke-induced inflammation. Therefore, we investigated the impact of G-CSF on cerebral DC migration and maturation as well as on the mediated immune response in an experimental stroke model in rats by means of transient middle cerebral artery occlusion (tMCAO). Immunohistochemistry and quantitative PCR were performed of the ischemic brain and flow cytometrical analysis of peripheral blood. G-CSF led to a reduction of the infarct size and an improved neurological outcome. Immunohistochemistry confirmed a reduced migration of DCs and mature antigen-presenting cells after G-CSF treatment. Compared to the untreated tMCAO group, G-CSF led to an inhibited DC activation and maturation. This was shown by a significantly decreased cerebral transcription of TLR2 and the DC maturation markers, CD83 and CD86, as well as by an inhibition of stroke-induced increase in immunocompetent DCs (OX62+OX6+) in peripheral blood. Cerebral expression of the proinflammatory cytokine TNF-α was reduced, indicating an attenuation of cerebral inflammation. Our data suggest an induction of DC migration and maturation under ischemic conditions and identify DCs as a potential target to modulate postischemic cerebral inflammation. Suppression of both enhanced DC migration and maturation might contribute to the neuroprotective action of G-CSF in experimental stroke.

Three cases of granulocyte colony-stimulating factor-producing urinary upper tract carcinomas

This report presents three cases of urinary upper tract carcinomas producing granulocyte colony-stimulating factor (G-CSF), with high blood leukocyte counts and poor prognoses. Case 1 was a 73-year-old man who underwent nephroureterectomy for left renal pelvic carcinoma. Pathologically, urothelial carcinoma (UC), high-grade, was observed, and immunohistochemical analysis showed positive staining for G-CSF. Progressive disease (PD) was observed despite administration of systemic chemotherapy for disease relapse, and the patient died 4.5 months after the operation. Case 2 was a 74-year-old man who had left renal pelvic carcinoma with para-aortic lymph-node metastases. The serum G-CSF was elevated (169 pg/ml). The patient refused any aggressive treatment, and died 2.3 months after his first visit to the hospital. Case 3 was a 75-year-old woman who had left renal pelvic carcinoma with adrenal metastasis. Biopsy confirmed the diagnosis as UC with squamous differentiation, and the serum G-CSF was elevated (138 pg/ml). Systemic chemotherapy was administered. However, the patient showed PD, and died 6.9 months after her first visit to the hospital. Effective treatment strategies are warranted for carcinomas producing G-CSF. Elucidation of the actions of G-CSF on both the carcinoma cells and the tumor microenvironment may contribute to the development of useful strategies.

Effects of interleukin-11 on the hematopoietic action of granulocyte colony-stimulating factor.

Interleukin-11 (IL-11, CAS 145941-26-0) is a cytokine that ameliorates thrombocytopenia induced by chemotherapy. Granulocyte colony-stimulating factor (G-CSF) is frequently used clinically as an adjuvant therapy to ameliorate neutropenia. In this study, it has been investigated whether IL-11 influences the hematopoietic action of G-CSF in vitro. IL-11 alone did not stimulate CFU-Meg colony formation in the absence of interleukin-3 (IL-3), but did in the presence of IL-3. However, IL-11 alone stimulated the formation of CFU-E, BFU-E and CFU-GM colonies, which were further enhanced by IL-3. G-CSF alone stimulated in a concentration dependent manner colony formation of CFU-GM and CFU-E, but not BFU-E or CFU-Meg. The addition of IL-11 enhanced the stimulatory effect of G-CSF on CFU-GM colony formation, and enabled G-CSF to stimulate colony formation of BFU-E, but not CFU-E. The addition of IL-3 further enhanced the stimulatory effect of G-CSF on CFU-E, BFU-E, and CFU-GM colony formation. However, G-CSF was unable to stimulate CFU-Meg colony formation for any of the cytokine combinations tested. These studies demonstrate that IL-11 has significant stimulatory effects on G-CSF-induced CFU-GM colony formation, suggesting that the therapeutic combination of the two growth factors could be beneficial for the treatment of myelosuppression induced by chemotherapy.

G-CSF and Erythropoietin Combination Therapy for Infarct Repair: Two Plus Two Equals Two?

G-CSF and Erythropoietin Combination Therapy for Infarct Repair: Two Plus Two Equals Two?

Long-term evaluation of granulocyte-colony stimulating factor on hypoxic-ischemic brain damage in infant rats

Hypoxia-ischemia (HI), as a major cause of fetal brain damage, has long-lasting neurological implications. Therefore, therapeutic interventions that attenuate the neuropathological outcome of HI while also improving the neurofunctional outcome are of paramount clinical importance. The aim of this study was to investigate the long-term functional and protective actions of granulocyte-colony stimulating factor (G-CSF) treatment in an experimental model of cerebral HI. Postnatal day-7 Sprague-Dawley rats were subjected to HI surgery, which entailed ligation of the right common carotid artery followed by 2 h of hypoxia (8% O(2)). Treatment consisted of subcutaneous injection of G-CSF at 1 h after hypoxia followed by an additional one injection per day for 5 days (6 total injections) or for 10 days (11 total injections). Animals were euthanized 5 weeks post-insult for extensive evaluation of neurological deficits and assessment of brain, spleen, heart, and liver damage. G-CSF treatment promoted somatic growth and prevented brain atrophy and underdevelopment of the heart. Moreover, reflexes, limb placing, muscle strength, motor coordination, short-term memory, and exploratory behavior were all significantly improved by both G-CSF dosing regimens. Long-term neuroprotection afforded by G-CSF in both morphological and functional parameters after a hypoxic-ischemic event in the neonate provides a rationale for exploring clinical translation.