Astrocyte-released cytokines induce ramification and outward K+ channel expression in microglia via distinct signalling pathways

Differentiation of microglial cells is characterized by transformation from ameboid into ramified cell shape and up-regulation of K+ channels. The processes of microglial differentiation are controlled by astrocytic factors. The mechanisms by which astrocytes cause developmental changes in morphological and electrophysiological properties of microglia have remained unclear. We show here that the cytokines transforming growth factor-beta (TGF-beta), macrophage colony-stimulating factor (M-CSF) and granulocyte/macrophage colony-stimulating factor (GM-CSF) are released by astrocytes at concentrations sufficient to induce ramification and up-regulation of delayed rectifier (DR) K+ channels in microglia. Transformation from ameboid into ramified morphology induced in microglia by exposure to astrocyte-conditioned medium (ACM) was inhibited by neutralizing antibodies against TGF-beta, M-CSF or GM-CSF, whilst ACM-induced DR channel expression was exclusively inhibited by antibodies against TGF-beta. Although both ramification and DR channel up-regulation occurred simultaneously, DR channel blockade by charybdotoxin failed to inhibit microglial ramification. The ACM-induced ramification of microglia was inhibited by the tyrosine kinase inhibitor genistein, whereas DR channel up-regulation did not occur in the presence of the serine/threonine kinase inhibitor H7. Our data suggest that astrocytes modulate processes of microglial differentiation in parallel but via distinct signalling pathways.     

GM-CSF and M-CSF modulate beta-chemokine and HIV-1 expression in microglia

Significant numbers of patients with acquired immunodeficiency syndrome (AIDS) develop CNS infection primarily in macrophages and microglial cells. Therefore, the regulation of human immunodeficiency virus type 1 (HIV-1) infection and activation of the brain mononuclear phagocytes subsequent to infection are important areas of investigation. In the current report, we studied the role of granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage-CSF (M-CSF) in the expression of antiviral beta-chemokines and HIV-1 p24 in cultures of primary human fetal microglia. We found that stimulation with GM-CSF or M-CSF induced macrophage inflammatory proteins (MIP-1alpha and MIP-1beta) and augmented RANTES expression, after HIV-1 infection of microglia. This was not due to the effect of GM-CSF on viral expression because GM-CSF was neither necessary nor stimulatory for viral infection, nor did GM-CSF enhance the expression of env-pseudotyped reporter viruses. Blocking GM-CSF-induced microglial proliferation by nocodazole had no effect on beta-chemokine or p24 expression. The functional significance of the GM-CSF-induced beta-chemokines was suggested by the finding that, in the presence of GM-CSF, exogenous beta-chemokines lost their anti-HIV-1 effects. We further show that although HIV-1-infected microglia produced M-CSF, they failed to produce GM-CSF. In vivo, GM-CSF expression was localized to activated astrocytes and some inflammatory cells in HIV-1 encephalitis, suggesting paracrine activation of microglia through GM-CSF. Our results demonstrate a complex interplay between CSFs, chemokines, and virus in microglial cells and may have bearing on the interpretation of data derived in vivo and in vitro.     

Effects of GM-CSF and ordinary supplements on the ramification of microglia in culture: A morphometrical study

Microglia transform from ameboid to ramified cells during development and display an ameboid appearance again under certain pathological conditions. Some cytokines produced by astrocytes may be responsible for the microglial transformation. In the present study, we compared the effects of cytokines, granulocyte/macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), and interleukin-3 (IL-3) on the morphology of rat cultured microglia. For quantitative evaluation, we employed “transformation index” as calculated by (perimeter of cell)²/4 π (cell area). GM-CSF facilitated the ramification of cultured rat microglia, which was effectively induced in a serum-free medium. However, M-CSF and IL-3 did not induce the ramification. A certain serum adhesion protein (possibly vitronectin) as well as other high molecular weight substances in fetal calf serum inhibited the GM-CSF-induced microglial ramification. Among ordinary supplements for a chemically defined medium, progesterone, insulin, and a high concentration of glucose suppressed the ramification. These findings suggest that GM-CSF may be involved in microglial ramification and that many kinds of supplements that are added to culture media profoundly affect the morphology of microglial cells. © 1996 Wiley-Liss, Inc.     

Differential regulation of microglial keratan sulfate immunoreactivity by proinflammatory cytokines and colony-stimulating factors

Resident microglia of the rat CNS express a unique type of keratan sulfate immunoreactivity (KS-IR) that is lacking on peripheral monocytes/macrophages and associated with a so far unknown proteoglycan core protein. Microglial KS-IR is downregulated during T-cell-mediated autoimmune inflammation but largely preserved in degenerative lesion paradigms. This study addresses the role of cytokines and colony-stimulating factors in the regulation of microglial KS-IR. In vitro, ramified microglia in coculture with astrocytes, but not isolated microglia, constitutively expressed KS-IR under control conditions. In both culture paradigms, KS-IR was increased significantly by macrophage- (M-CSF) and granulocyte/macrophage colony-stimulating factors (GM-CSF), as well as tumor necrosis factor-alpha (TNF-alpha). By contrast, the Th1 cytokine interferon-gamma (IFN-gamma) downregulated KS-IR, both when applied alone or in combination with either GM-CSF, M-CSF, or TNF-alpha. In vivo, the intracerebroventricular administration of IFN-gamma, but not TNF-alpha, to healthy rats led to an almost complete disappearance of KS-IR from ramified brain microglia. Our data suggest that the expression of microglial KS-IR is under dominant negative control by the Th1 cell cytokine IFN-gamma and represent the first evidence of cytokine-dependent proteoglycan regulation in the CNS.     

GM-CSF promotes proliferation of human fetal and adult microglia in primary cultures

Proliferation of microglia/macrophages is a common finding in many central nervous system diseases. To identify mitogenic signals for human microglia, we examined primary cultures of human fetal and adult microglia after stimulation with cytokines, colony stimulating factors (CSFs), or LPS, using proliferating cell nuclear antigen (PCNA) expression as an index of cell proliferation. The results showed that both M-CSF and GM-CSF induced microglial proliferation in fetal and adult human cultures, but that GM-CSF provided a much stronger stimulus. At 96 h post-stimulation, the mean PCNA labeling index was 2.4 for M-CSF and 13.3 for GM-CSF in fetal microglia; in adult microglia, the PCNA labeling index was 4.7 for M-CSF and 9.0 for GM-CSF. The effect of GM-CSF on fetal microglia was dose dependent and synergistic with M-CSF. LPS abolished the basal level of PCNA labeling in adult microglia, but in fetal microglia, caused a slight increase in PCNA labeling (1.9) at 96 h and consistently enhanced microglial cell survival and differentiation into highly branched cells. The production of GM-CSF in purified human fetal astrocyte and microglial cultures was examined after stimulation with LPS, TNF-α, or IL-1β. Unlike M-CSF, neither cell type produced GM-CSF in unstimulated cultures; however, when stimulated with IL-1β, astrocytes expressed GM-CSF mRNA and protein, which accumulated in the culture through 72 h. In microglia, LPS was the only effective inducing agent. An immunocytochemical study performed to identify in vivo sources of GM-CSF revealed selective labeling of reactive astrocytes in active lesions of multiple sclerosis and senile plaques of Alzheimer's disease. Our data demonstrate that both fetal and adult human microglia are capable of proliferation in response to CSFs, GM-CSF being the more effective stimulus.     

Colony-stimulating factors in rapid eye movement sleep and non-rapid eye movement sleep regulation

Although several cytokines are known to be somnogenic, no study has been conducted to examine whether colony-stimulating factors (CSF) affect sleep. Therefore, we studied the effects of granulocyte-macrophage CSF (GM-CSF) and macrophage CSF (M-CSF) on sleep in rats and their possible mechanism of action. At the dose of 10 pmol, GM-CSF or M-CSF significantly increased both non-rapid eye movement and rapid eye movement (REM) sleep or REM sleep only when infused intracerebroventricularly during the dark period. When injected locally in the hypothalamus, GM-CSF and M-CSF increased nitric oxide (NO) production. Thus, NOergic neural signals in the hypothalamus may take part in the somnogenic action of CSF.     

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.     

Macrophage colony stimulating factor is a crucial factor for the intrinsic macrophage response in mice heterozygously deficient for the myelin protein P0

Mouse mutants heterozygously deficient for the myelin protein P0 (P0+/-) resemble certain forms of human hereditary neuropathies. Endoneurial macrophages of intrinsic origin are intimately involved in the pathogenesis of the demyelinating neuropathy in these mutants. We have previously shown that deficiency for macrophage colony stimulating factor (M-CSF) prevents an increase of the number of endoneurial macrophages and alleviates the mutants' demyelinating phenotype. The aim of this study was to investigate which population of endoneurial macrophages - long-term resident macrophages or recently infiltrated macrophages - is affected by M-CSF deficiency. For this purpose, we generated bone marrow chimeric mice by transplanting GFP+ bone marrow into P0 mutants (P0+/-) and P0 mutants that lack M-CSF (P0+/- mcsf-op). This enabled us to discriminate recently infiltrated short-term resident GFP+ macrophages from long-term resident GFP- macrophages. Three months after bone marrow transplantation, P0+/- mice expressing M-CSF showed a substantial upregulation and activation of both GFP- and GFP+ macrophages in femoral nerves when compared to P0+/+ mice. In contrast, in P0+/- mcsf-op mutants, both GFP- and GFP+ macrophages did not substantially increase. Only small numbers of GFP+ but no GFP- macrophages were activated and phagocytosed myelin in chimeric P0+/- mcsf-op mutants, possibly reflecting recent activation outside the endoneurium before entering the nerve. Our findings demonstrate that M-CSF is crucial for the activation, in situ increase and myelin phagocytosis of both long-term and short-term resident endoneurial macrophages in P0+/- myelin mutants. M-CSF is, therefore, considered as a target candidate for therapeutic strategies to treat human demyelinating neuropathies.     

Granulocyte-macrophage colony stimulating factor inhibits class II major histocompatibility complex expression and antigen presentation by microglia

Granulocyte-macrophage colony stimulating factor (GM-CSF) modulates various functions of monocytes/ macrophages including antigen-presenting capacity. Recently it was found that astrocytes produce GM-CSF in the central nervous system (CNS) and that GM-CSF can induce proliferation and morphological changes of microglia. Here we show that GM-CSF can down regulate the interferon-γ-mediated induction of major histocompatibility complex (MHC) class II antigens in microglia, but not in astrocytes. GM-CSF pretreatment completely prevents myelin basic protein-specific T cell proliferation induced by microglia but not astrocytes. GM-CSF did not affect the cell surface expression on microglia of either MHC class I or cell adhesion molecules. The inhibition of microglial MHC class II expression and antigen-presenting function is specific for GM-CSF, as treatment with a different CSF (interleukin-3) did not modulate microglial phenotype or functional capacity. These data suggest that GM-CSF might be involved in the regulation of immune responses within the central nervous system.     

Trophic effect of erythropoietin and other hematopoietic factors on central cholinergic neurons in vitro and in vivo

In vitro granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), erythropoietin (EPO), and erythroid differentiation factor (EDF) augmented choline acetyltransferase (ChAT) activity in mouse embryonic primary septal neurons and in cholinergic hybridoma cell line, SN6.10.2.2. This is similar to the effects seen with interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Moreover, in vivo GM-CSF and EPO promoted survival of septal cholinergic neurons in adult rats which had undergone fimbria-fornix transections. These results suggest that some of the hematopoietic factors act on cholinergic neurons as ‘neurotrophic factors’ to influence the differentiation, maintenance and regeneration of these neurons.     

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.     

Colony-stimulating factors regulate programmed cell death of rat microglia/brain macrophages in vitro

Programmed cell death of activated microglia appears to be one mechanism how steady state of microglia is achieved in vivo. Programmed cell death of microglia might result either from the downregulation of microglial mitogens/survival factors or from signals which directly induce microglial cell death. To further elucidate the mechanisms regulating programmed cell death in microglia, growth factor and cytokine dependence of microglial proliferation and cell death have been examined in vitro in microglia/brain macrophage cultures established from neonatal rat brain. Microglial proliferation was assessed by PCNA labelling and DNA fragmentation by the TUNEL technique in the presence or absence of several cytokines including IL-1, IL-6, TGFβ1, TNFα, M-CSF and GM-CSF. Results of TUNEL labellings were supplemented by gel electrophoretic analysis of DNA extracted from cultured microglia which showed laddering of DNA fragments. Of all cytokines/growth factors tested, GM-CSF and M-CSF were not only the strongest microglial mitogens but, moreover, withdrawal of M-CSF or GM-CSF significantly enhanced rates of microglial cell death by DNA fragmentation. Expression of microglial growth factors, in particular colony-stimulating factors, may thus be instrumental in controlling steady states of microglia in the injured nervous system.     

M-CSF production by HIV-1-infected monocytes and its intrathecal synthesis implications for neurological HIV-1-related disease

Macrophage-colony stimulating factor (M-CSF) is detectable in the cerebrospinal fluid (SF) of HIV-1-infected patients, and may be produced intrathecally by both reactive astrocytes and cells of the monocyte/macrophage (MO) lineage, microglial cells included. Since MO constitute the target cells for HIV-1 in the central nervous system (CNS), the culture conditions that induce M-CSF production by HIV-1-infected MO were studied. MO cultures infected with supernatants (SN) of HIV-1-infected peripheral blood lymphocyte (PBL) cultures produced only trace or undetectable amounts of M-CSF. Co-cultures of MO with normal PBL released high amounts of M-CSF, suggesting that viable cell-to-cell interactions are required to induce cytokine production by MO and/or PBL. M-CSF production was markedly increased in the MO co-cultured with HIV-1-infected PBL, thus implying that HIV-1 induces increased cytokine synthesis/release by MO and/or PBL only when cell membrane-associated messages are operating. Intracerebrally synthesized M-CSF by HIV-1-infected MO may play a role in promoting viral replication/spread within the CNS, and inducing brain damage by stimulating microglia proliferation, and neurotoxic factor release by these cells.     

Macrophage colony stimulating factor (M-CSF) protects spiral ganglion neurons following auditory nerve injury: morphological and functional evidence

Because hearing disturbance due to auditory nerve dysfunction imposes a formidable burden on human beings, intense efforts have been expended in experimental and clinical studies to discover ways to restore normal hearing. However, the great majority of these investigations have focused on the peripheral process side of bipolar auditory neurons, and very few trials have focused on ways to halt degenerative processes in auditory neurons from the central process side (in the cerebellopontine angle). In the present study, we investigated whether administration of macrophage colony-stimulating factor (M-CSF) could protect auditory neurons in a rat model of nerve injury. The electrophysiological and morphological results of our study indicated that M-CSF could ameliorate both anterograde (Wallerian) and retrograde degeneration in both the CNS and PNS portions of the auditory nerve. We attribute the success of M-CSF therapy to the reported functional dichotomy (having the potential to cause both neuroprotective and neurotoxic effects) of microglia and macrophages. Whether the activities of microglia/macrophages are neuroprotective or neurotoxic may depend upon the nature of the stimulus that activates the cells. In the present study, the neuroprotective effects of M-CSF that were observed could have been due to M-CSF we administered and to M-CSF released from endothelial cells, resident cells of the CNS parenchyma, or infiltrating macrophages. Another possibility is that M-CSF ameliorated apoptotic auditory neuronal death, although this hypothesis remains to be proved in future studies.     

M-CSF deficiency leads to reduced metallothioneins I and II expression and increased tissue damage in the brain stem after 6-aminonicotinamide treatment

6-Aminonicotinamide (6-AN) is a niacin antagonist, which leads to degeneration of gray-matter astrocytes followed by a vigorous inflammatory response. Macrophage colony stimulating factor (M-CSF) is important during inflammation, and in order to further clarify the roles for M-CSF in neurodegeneration and brain cell death, we have examined the effect of 6-AN on osteopetrotic mice with genetic M-CSF deficiency (op/op mice). The 6-AN-induced degeneration of gray-matter areas was comparable in control and op/op mice, but the numbers of reactive astrocytes, macrophages, and lymphocytes in the damaged areas were significantly decreased in op/op mice relative to controls. The levels of oxidative stress (as determined by using immunoreactivity for inducible nitric oxide synthase, nitrotyrosine, and malondialdehyde) and apoptotic cell death (as determined by using TUNEL and immunoreactivity for caspases and cytochrome c) were significantly increased in 6-AN-injected op/op mice relative to controls. From a number of antioxidant factors assayed, only metallothioneins I and II (MT-I+II) were decreased in op/op mice in comparison to controls. Thus, the present results indicate that M-CSF is an important growth factor for coping with 6-AN-induced central nervous system damage and suggest that MT-I+II are likely to have a significant role.     

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.     

Exploring the relationship of macrophage colony-stimulating factor levels on neuroaxonal metabolism and cognition during chronic human immunodeficiency virus infection

Macrophage colony-stimulating factor (M-CSF) promotes macrophage differentiation, increases susceptibility of macrophages to viral infection, and enhances human immunodeficiency virus (HIV) replication in infected macrophages. Given the current model of HIV neuropathogenesis, which involves monocyte trafficking into the central nervous system, immune factors linked with macrophage maturation and survival may be associated with cognitive decline (measured by neuropsychological z-score [NPZ-8] or Memorial Sloan-Kettering [MSK] score) and alterations in a marker of neuronal integrity,N-acetylaspartate (NAA). Fifty-four chronically infected HIV+ subjects underwent neuropsychological assessment, magnetic resonance spectroscopic imaging, and quantification of M-CSF in plasma and cerebrospinal fluid (CSF) at baseline. Thirty-nine of those subjects underwent further examination at 3 and 10 months after initiation of combination antiretroviral therapy (ART) regimens. Within 3 months of therapy use, CSF M-CSF and viral RNA levels were reduced, whereas NAA concentrations in many brain regions were increased. Neither baseline levels nor the change in M-CSF levels had the ability to predict changes in NAA levels observed after 10 months of combination ART use. At study entry those with the lowest M-CSF levels in the CSF had the least cognitive impairment (NPZ-8). Those who had higher baseline CSF MCSF levels and exhibited larger decreases in M-CSF after therapy, tended to have greater cognitive improvement after 10 months. Increased prevalence of M-CSF in the setting of HIV infection could contribute to neuronal injury and may be predictive of cognitive impairment.     

Effects of GM-CSF on the neural progenitor cells

Granulocyte macrophage colony stimulating factor (GM-CSF) is a potent hematopoietic cytokine, which stimulates stem cell proliferation in the bone marrow. We now report that GM-CSF receptors expressed on neural progenitor cells and can mediate a biological response in cells to treat with GM-CSF treated neural progenitor cells exhibited a proliferative response and a marked decrease in terminal differentiation to mature neuron or astrocytes. GM-CSF treatment also suppressed neural progenitor cell apoptosis. These findings suggest that GM-CSF can stimulate the proliferation and inhibit the apoptosis of neural progenitor cells to expand the progenitor population, and that GM-CSF has a potential role in neural development or repair.