Increase of macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor receptors in the regenerating rat facial nucleus

Proliferation of microglial cells commonly occurs in the response of the central nervous system to injury, but little is known about how this process is regulated in vivo. Here we have studied the expression of receptors to macrophage colony-stimulating factor (MCSF) and granulocyte-macrophage colony-stimulating factor (GMCSF) in the normal and regenerating rat facial motor nucleus using receptor immunocytochemistry and in situ ligand binding methods. Under normal conditions, immocytochemical staining with anti-MCSF receptor (MCSFR) antibody revealed a moderate but selective labelling of microglia-like cells of the facial motor nucleus. This immunostaining also colocalized with MUC102, a new monoclonal antibody raised against microglial cells in the rat central nervous system. Axotomy of the facial nerve led to a rapid increase in MCSFR-staining intensity 1 day after injury, which became maximal 7 days postoperatively and then decreased. A similar but somewhat slower increase was also observed for the specific [¹²⁵I]MCSF binding wiht a maximum at 7 days Specific [¹²⁵I]GMCSF binding also increased, peaking at 4 days postoperatively and then rapidly decreasing to normal levels at 21 days after axotomy. In summary, axotomy of the facial nerve led to a rapid increase in receptors for MCSF and GMCSF, which coincided with the pattern of microglial proliferation in the regenerating facial motor nucleus. This apparent up-regulation of receptors for microglial growth factors may play an important role in preparing the microglia to participate in the cellular response to injury in the regenerating central nervous system.     

Functional dichotomy of mouse microglia developed in vitro: Differential effects of macrophage and granulocyte/macrophage colony-stimulating factor on cytokine secretion and antitoxoplasmic activity

After differentiation either with exogenous macrophage (M) or with granulocyte/macrophage (GM) colony-stimulating factor (CSF) microglial cells were isolated from neonatal mouse brain cell cultures and were comparatively tested for secretory immune effector cell functions. Both factors obviously do not promote the development of cells with biased growth requirement; however, the two microglia populations displayed distinct potentials to produce inflammatory cytokines. Upon gradual stimulation by lipopolysaccharide, the cells harvested from M-CSF-driven culture released more interleukin-1 and tumor necrosis factor activity, GM-CSF-grown cells on the contrary proved superior in interleukin-6 secretion. This pattern was paralleled by correspondingly different kinetics of cytokine release in both types of microglial cells. When infected with Toxoplasma gondii only GM-CSF-differentiated cells were able to restrict the intracellular multiplication of tachyzoites in the absence of external stimuli. As described for interferon-γ-treated macrophages, the antiparasitic activity of this microglia population is due to the synthesis of reactive nitrogen intermediates, since it was antagonized by N^G-monomethyl-L-arginine, a competitive inhibitor of the arginine-dependent metabolic pathway. Complementary to previous data which attest an intrinsic capability for antigen presentation to GM-CSF-grown microglia, the functional state of the cells elicited by M-CSF and GM-CSF, respectively, may correspond to the resting and an activated form of microglia as distinguished in vivo.     

Granulocyte macrophage colony-stimulating factor treatment of a patient in myasthenic crisis: Effects on regulatory T cells

Introduction: In this study we describe a patient with a prolonged myasthenic crisis refractory to conventional immunomodulatory therapy who was treated with GM-CSF (granulocyte macrophage colony-stimulating factor, sargramostim). Methods: T-regulatory cell (Treg) suppressive function and Foxp3 expression were evaluated before and after treatment with GM-CSF. Results: Treatment with GM-CSF was associated with clinical improvement, expansion in the circulating numbers of Foxp3(+) cells, increase in Foxp3 expression levels in Tregs, early improvement in Treg suppressive capacity for AChR-α-induced T-cell proliferation, and subsequent enhancement in Treg suppression of polyclonal T-cell proliferation. Conclusion: Although definitive conclusions cannot be drawn from a single case, the correlation with similar findings in GM-CSF-treated animals with experimental autoimmune myasthenia gravis suggests further exploration of the effects of GM-CSF in myasthenia gravis should be studied in a clinical trial setting. Muscle Nerve 46: 449-453, 2012.     

Circulating macrophage colony-stimulating factor levels and stroke: A Mendelian randomization study

ObjectivesColony-stimulating factor 1 (CSF1), also known as macrophage colony-stimulating factor, has been shown to be associated with risk of ischemic stroke in conventional epidemiological study. We performed a Mendelian randomization analysis to evaluate the effects of genetically predicted circulating CSF1 levels on stroke and carotid intima-media thickness (cIMT).MethodsGenetic variants robustly associated with CSF1 levels, located in the vicinity of the CSF1 gene (cis), were used as instruments for CSF1 levels. Genetic association estimates for ischemic stroke and its subtypes, intra-cerebral hemorrhage (ICH), and cIMT were obtained from MEGASTROKE (60,341 cases and 454,450 controls), ISGC (1,545 cases and 1,481 controls), and UK Biobank (22,179 individuals), respectively.ResultsGenetically predicted higher CSF1 levels was significantly associated with a higher risk of any ischemic stroke, large artery stroke (LAS) and cardioembolic stroke (CES), but not with small vessel stroke (SVS) and ICH. The odds ratios (ORs) per genetically predicted one standard deviation (SD) increase in circulating CSF1 levels were 1.11 (95% CI 1.04–1.17) for any ischemic stroke, 1.23 (95% CI 1.07–1.42) for LAS, 1.18 (95% CI 1.05–1.33) for CES, 1.07 (95% CI 0.94–1.21) for SVS, and 1.15 (95% CI 0.73–1.83) for ICH. Similarly, we also found that genetically predicted higher CSF1 levels were associated with higher cIMT, as a measure of subclinical atherosclerosis (cIMT, β 0.016, 95% CI, 0.004-0.029).ConclusionsThis study provides evidence that genetically predicted higher CSF1 levels was associated with higher risk of any ischemic stroke, LAS, and CES. Whether targeting CSF1 or its receptors can reduce the risk of ischemic stroke needs further study.     

Microglia in colony-stimulating factor 1-deficient op/op mice

Mice that are homozygous for the autosomal recessive mutation osteopetrosis (op) suffer from a general skeletal sclerosis, and the numbers of macrophages in various tissues are significantly decreased. We report that microglia in op/op mice are not affected by the mutation. They have normal morphology and are present in the CNS in normal frequency. In cultures, disaggregated cells of neopallia can form microglia, but such cells from neopallia of op/op mice form microglia only when colony-stimulating factor 1 (CSF-1) is added to the culture medium. The addition of granulocyte/macrophage (GM)-CSF or interleukin (IL)-3 to the culture medium does not stimulate production of microglia. Microglia that form in op/op neopallial cell cultures, in the presence of CSF-1, are capable of Fc-receptor-mediated phagocytosis. Based on our experiments, it seems that microglia are CSF-1 dependent but in op/op mice (in which CSF-1 is absent) microglia may use other locally produced factors. © 1995 Wiley-Liss, Inc.     

银屑病患者骨髓基质细胞分泌干细胞因子和粒细胞集落刺激因子的水平

背景：课题组在前期研究中发现银屑病患者骨髓造血细胞活性存在异常。 目的：观察银屑病患者骨髓基质细胞分泌干细胞因子和粒细胞集落刺激因子的水平。 设计、时间及地点：病例-对照观察,于2007-10/2008-08在太原市中心医院皮肤科实验室完成。 对象：选择太原市中心医院皮肤科临床及病理确诊为寻常型银屑病的门诊患者24例,男16例,女8例;年龄16~59岁;另取血液科骨穿后经筛选的正常骨髓20例作为对照组,性别、年龄与银屑病患者匹配。 方法：采用密度梯度离心法分离银屑病患者与对照组骨髓单一核细胞,通过贴壁法培养骨髓基质细胞,收集传了3代后又培养72 h的骨髓基质细胞及培养上清。 主要观察指标：流式细胞仪检测细胞表面标志并用ELISA法检测上清液中干细胞因子和粒细胞集落刺激因子的水平。 结果：流式细胞仪检测结果显示,90%以上细胞表面抗原高表达CD29,而CD34、CD45及HLA-DR表达阴性,即骨髓基质细胞纯度在90%以上。银屑病组骨髓基质细胞培养上清液的干细胞因子和粒细胞集落刺激因子表达均显著高于对照组 (P < 0.01)。 结论：银屑病患者骨髓基质细胞分泌干细胞因子和粒细胞集落刺激因子存在异常,提示银屑病患者骨髓造血微环境发生了改变。     

Trophic effect of granulocyte-macrophage colony-stimulating factor on central cholinergic neurons in vitro

Granulocyte-macrophage colony-stimulating factor (GM-CSF) elevated choline acetyltransferase (ChAT) activities of mouse septal neurons as well as of cholinergic hybridoma line cells SN6.10.2.2 in vitro. It augmented ChAT activities and neurite extension of interleukin 3-activated cholinergic neurons. Thus, GM-CSF should be added as a trophic factor for central cholinergic neurons.     

Granulocyte macrophage colony stimulating factor stimulates in vitro proliferation of astrocytes derived from simian mature brains

In the brain, granulocyte-macrophage colony stimulating factor (GM-CSF) may be released by infiltrated cells of the immune system including T and B lymphocytes and mononuclear phagocytes, but also by nervous system resident cells such as microglia and astrocytes. Astrocyte-secreted GM-CSF may play an important role in enhancing the local inflammatory response to central nervous system (CNS) injury and in recruting microglia and activated macrophages. In this study, we demonstrated that GM-CSF, as TNFα and IL 6, stimulates in vitro proliferation of simian astrocytes in primary cultures. Results were confirmed by blocking experiments performed with a specific neutralizing mAb directed against GM-CSF. Furthermore, we demonstrated that GM-CSF mediates its effect on these cells through the α subunit of the GM-CSF receptor which is constitutively expressed at the membrane of the cultured simian astrocytes as assessed by immunofluorescence. GM-CSF effects on astrocytes could be involved in astrocytosis, a hallmark of various neurological injuries and in inflammatory processes in an autocrine manner. © 1996 Wiley-Liss, Inc.     

The relative number of macrophages/microglia expressing macrophage colony-stimulating factor and its receptor decreases in multiple sclerosis lesions

The activation of macrophages/microglia in multiple sclerosis (MS) lesions plays a central role in the effector phase of myelin breakdown. The precise patterns of macrophage/microglia activation during demyelination have not yet been defined. The growth and activating factor macrophage-colony stimulating factor (M-CSF) and its specific receptor (M-CSFR) may be involved in this process. The present study investigated the expression of M-CSF and M-CSFR mRNA by in situ hybridization in 60 lesions from 32 MS patients. In the control and periplaque white matter, microglia was almost completely M-CSFR positive. Irrespective of the demyelinating activity, an increased number of cells expressed M-CSF or M-CSFR mRNA within the lesions. However, despite the tremendous increase in macrophages/microglia within the lesions, the relative number of these cells expressing M-CSF or M-CSFR decreased. There was no correlation of M-CSF or M-CSFR expression with active myelin breakdown. The correlation between the clinical course and the expression of M-CSF or M-CSFR mRNA revealed significant differences with the lowest expression in primary progressive MS. These results suggest a downregulation of M-CSF and M-CSFR inside the MS plaque probably due to the high amount of macrophage-derived cytokines or mediators. Nevertheless, the differences in the relative number of cells expressing the M-CSF/M-CSFR pathway implicate that this pathway may be an important contributory factor in different forms of MS pathology.     

mdx小鼠与C57小鼠骨髓基质细胞体外分化能力的比较**

背景：肌营养不良症是一种渐进性致死性X连锁隐性遗传性肌肉疾病,目前无特效治疗。肌营养不良症模型鼠(mdx小鼠)的骨髓基质细胞增殖及定向分化能力是否正常,自身骨髓移植是否合适还有待研究。 目的：观察mdx小鼠骨髓基质细胞体外培养时的增殖及多向分化能力。 方法：取mdx小鼠与C57小鼠胫股骨骨髓基质细胞体外培养,经吉姆萨染色后观察其形成成纤维细胞集落形成单位的能力;通过不同诱导液使骨髓基质细胞定向分化为成骨、成脂、成肌细胞,观察其形态学特性;并分别用Von kossa 染色、油红O染色、免疫荧光检测desmin阳性细胞对已分化细胞进行鉴定和分化率比较;培养1周时,提取分化细胞总RNA,反转录后,用real-time PCR检测各分化细胞相关基因表达。 结果与结论：mdx小鼠骨髓基质细胞形成的成纤维细胞集落形成单位数目和体积均小于C57小鼠。其成骨、成肌分化的效率均明显低于C57小鼠(P < 0.01),两种小鼠的骨髓基质细胞成脂分化效率差异无显著性(P > 0.05)。real-time PCR检测结果显示,与C57小鼠相比,mdx小鼠的骨髓基质细胞成骨、成肌基因表达均有不同程度下降,而成脂基因表达无明显差异。结果提示,mdx小鼠的骨髓基质细胞体外培养时的增殖及定向分化能力较C57小鼠下降,与Dystrophin 基因缺失有关,mdx小鼠自体骨髓移植将会受限。     

Enhanced regeneration in spinal cord injury by concomitant treatment with granulocyte colony-stimulating factor and neuronal stem cells.

Granulocyte colony-stimulating factor (G-CSF) inhibits programmed cell death and stimulates neuronal progenitor differentiation. Neuronal stem cells transplanted into injured spinal cord can survive, differentiating into astroglia and oligodendroglia, and supporting axon growth and myelination. Herein, we evaluate the combined effects of G-CSF and neuronal stem cells on spinal cord injury. For 40 Sprague-Dawley rats (n=10 in each group) transverse spinal cord resections at the T8-9 level were carried out, leaving an approximately 2-mm gap between the distal and proximal ends of the cord. Neuronal stem cells embedded in fibrin glue treated with or without G-CSF (50 microg/kg x 5 days) (groups III and IV) or fibrin glue with or without G-CSF (50 microg/kg x 5 days) (groups I and II) were transplanted into the gap in the injured spinal cord. Spinal cord regeneration was assessed using a clinical locomotor rating scale scores and electrophysiological, histological and immunohistochemical analysis 3 months after injury. Regeneration was more advanced in group IV than in groups III or II according to the clinical motor score, motor evoked potential, and conduction latency. Most advanced cord regeneration across the gap was observed in group IV rats. Higher densities of bromodeoxyuridine in the injured area and higher expression levels of Neu-N and MAP-2 over the distal end of the injured spinal cord were observed in group IV compared with groups II or III, but there was no significant difference in expression of glial fibrillary acid protein. This synergy between G-CSF and neuronal stem cells may be due to increased proliferation of progenitor cells in the injured area and increased expression of neuronal stem cell markers extrinsically or intrinsically in the distal end of injured cord.     

骨髓干细胞移植治疗Duchenne型肌营养不良鼠的实验研究

目的　研究骨髓干细胞移植治疗Duchenne型肌营养不良鼠 (mdx鼠 )的效果。方法　取 4～ 5周龄昆明鼠骨髓干细胞 ,体外培养 3d ,静脉移植到 7Gyγ射线预处理 7～ 8周龄mdx鼠体内。临床监测受体鼠移植物抗宿主病 (GVHD)表现 ,并对移植 12周mdx鼠的运动功能、肌电生理、dystrophin蛋白表达情况进行检测。 结果　 5只 7Gy剂量放疗mdx鼠 ,静脉移植 4 8× 10 6骨髓干细胞 ,3个月后 ,肌电图指标有了部分改善 ;10 %肌纤维表达了dystrophin蛋白。结论　静脉移植同种非同系鼠骨髓干细胞治疗mdx鼠有效 ,显示骨髓干细胞移植治疗DMD有着理想的前景。     

Grafted neural stem cells increase the life span and protect motoneurons in pmn mice

In this study, we have grafted neural stem cells (NSCs) into the lumbar spinal cord of a mouse mutant that has a specific loss of motoneurons (progressive motor neuronopathy/pmn). A small number of grafted cells ( approximately 3000) increased the life span of the mice by 56%. The improved survival was accompanied by a rescue of host motoneurons, a stabilization in the weight and an increase in the size of the muscle fibers. The grafted NSCs were small and round and exhibited no neural markers, suggesting that they remained in an undifferentiated state. Thus grafting of NSCs in a mouse model with motoneuron degeneration exerts a neuroprotective effect.     

Granulocyte colony-stimulating factor attenuates neuronal death and promotes functional recovery after spinal cord injury in mice

Granulocyte colony-stimulating factor (G-CSF) is a protein that stimulates differentiation, proliferation, and survival of granulocytic lineage cells. Recently, a neuroprotective effect of G-CSF was reported in a model of cerebral infarction. The aim of the present study was to elucidate the potential therapeutic effect of G-CSF for spinal cord injury (SCI) in mice. We found that G-CSF is neuroprotective against glutamate-induced cell death of cerebellar granule neurons in vitro. Moreover, we used a mouse model of compressive SCI to examine the neuroprotective potential of G-CSF in vivo. Histologic assessment with cresyl violet staining revealed that the number of surviving neurons in the injured spinal cord was significantly increased in G-CSF-treated mice. Immunohistochemistry for neuronal apoptosis revealed that G-CSF suppressed neuronal apoptosis after SCI. Moreover, administration of G-CSF promoted hindlimb functional recovery. Examination of signaling pathways downstream of the G-CSF receptor suggests that G-CSF might promote functional recovery by inhibiting neuronal apoptosis after SCI. G-CSF is currently used in the clinic for hematopoietic stimulation, and its ongoing clinical trial for brain infarction makes it an appealing molecule that could be rapidly placed into trials for patients with acute SCI.     

Role of macrophage migration inhibitory factor in primary glioblastoma multiforme cells

Macrophage migration inhibitory factor (MIF) is a protein that is overexpressed in many tumors, such as colon and prostate cancer, melanoma, and glioblastoma multiforme (GBM). In its function as a cytokine, MIF induces angiogenesis, promotes cell cycle progression, and inhibits apoptosis. Recently, the molecular signal transduction has been specified: MIF has been found to be a ligand to the CD74/CD44-receptor complex and to activate the ERK1/2 MAPK cascade. In addition MIF binds to the chemokine receptors CXCR2 and CXCR4. This effects an integrin-dependent leukocyte arrest and mediates leukocyte chemotaxis. Recent work has described a clearer role of MIF in GBM tumor cell lines. The current study used human primary GBM cells. We show that inhibition of MIF with ISO-1, an inhibitor of the D-dopachrome tautomerase site of MIF, reduced the growth rate of primary GBM cells in a dose-dependent manner, and in addition ISO-1 increased protein expression of MIF and its receptors CD74, CXCR2, and CXCR4 in vitro but decreased expression of CD44. Furthermore, hypoxia as cell stressor increases the protein expression of MIF in primary GBM cells. These results underscore the importance of MIF in GBM and show that MIF and its receptors may be a promising target for the treatment of malignant gliomas.     

Cells over-expressing EAAT2 protect motoneurons from excitotoxic death in vitro

Amyotrophic lateral sclerosis is an incurable disease in which cerebral and spinal motoneurons degenerate, causing paralysis and death within 2-5 years. One of the pathogenic factors of motoneuron death is a chronic excess of glutamate, which exceeds its removal by astrocytes, i.e. excitotoxicity. Extra glutamate uptake in the spinal cord may slow down or prevent motoneuron death. We have engineered cells over-expressing the main glutamate transporter and tested their potential to rescue motoneurons exposed to high levels of glutamate in vitro. The engineered cells protected motoneurons in a motoneuron-astrocyte co-culture at glutamate concentrations when astrocytes were no longer capable of removing glutamate. This suggests that engineered cells, introduced into the spinal column, can help remove glutamate, thereby preventing motoneuron death.     

Electrophysiological properties of in vitro hippocampal pyramidal cells from normal and staggerer mutant mice

Electrophysiological properties of intracellularly recorded CA1 pyramidal cells from normal and staggerer mice were compared by using hippocampal slices maintained in vitro. In staggerer mice, the passive membrane properties of these neurons as well as their synaptic potentials elicited by stratum radiatum stimulation were very similar to those observed in normal mice. In control and mutant mice and in standard Krebs solution, CA1 pyramidal cells mainly fired tetrodoxin (TTX)-sensitive fast spikes but could also generate slow spikes. In both groups, replacement of calcium (Ca) by barium (Ba) or introduction of TEA in the bathing medium prolonged the repolarization of the fast spikes and suppressed the brief spike afterhyperpolarization which normally followed them, thus suggesting that both events involve fast potassium conductances. Furthermore, in both groups of animals, TEA and Ba enhanced the slow spikes and induced the appearance of prolonged depolarizations. These slow events were TTX-resistant and were abolished by the Ca channel blockers cadmium or cobalt, thus suggesting that they are Ca-dependent. On the whole, the present results indicate that the staggerer mutation which yields marked abnormalities in the bioelectrical properties of cerebellar Purkinje cells has no such effect on CA1 pyramidal cells.     

In vitro stimulation of glia cells by a lymphocyte-produced factor

The factors responsible for the activation of astrocytes surrounding inflammatory brain tissues are unknown. The present study was designed to examine the ability of lymphocytes to produce astrocyte stimulating activity in cell culture. Normal rat lymphocytes stimulated with Concanavalin A, or sensitized lymphocytes, challenged with antigen in vitro, activate cultured rat glia cells by a soluble mediator which we have termed glia stimulating factor (GSF). In undifferentiated glioblasts both RNA synthesis, as measured by [5-3H]uridine uptake, and DNA synthesis, as measured by [6-3H]thymidine uptake, were stimulated by the presence of GSF. Preliminary characterisation showed the GSF to be non-dialysable and heat stable at 56 degrees C for 30 min, but not stable at 80 degrees C for 30 min. To study the effect of this factor on differentiated glia cells, brain cell cultures were treated with dibutyryl cyclic AMP (db-cAMP) which induces a morphologic transformation of glioblasts to multipolar cells that have a characteristic astrocytic appearance. After addition of GSF to db-cAMP treated astrocytes only an increase in RNA synthesis was observed. The significance of this in vitro phenomenon, mediated by a glia stimulating factor, to activation of astrocytes and astrocytic gliosis in human brain diseases is discussed.