Chemokines induce migration and changes in actin polymerization in adult rat brain microglia and a human fetal microglial cell line in vitro

Microglia, the resident macrophages of the central nervous system, are the primary cells to respond to injury in the brain, both in inflammation, e.g., in multiple sclerosis, and trauma. Chemokines are potential mediators of microglial cell recruitment to sites of injury; thus, the ability of microglia to migrate in response to a number of chemokines was assessed. The chemokines monocyte chemoattractant protein 1, macrophage inflammatory protein 1α, macrophage inflammatory protein 1β, RANTES (regulated upon activation normal T cell expressed and secreted), interleukin 8, and IP‐10 (interferon gamma inducible protein‐10), induce migration and changes in the distribution of f‐actin in adult rat microglia and a human microglial cell line, CHME3, in vitro. Both cell types show a significant migration response, above control levels, to all the chemokines tested in a typical dose‐dependent manner. These chemokines also induced a reorganization of the actin cytoskeleton of the cells. This study indicates that chemokines play an important role in the recruitment of microglia to areas of central nervous system inflammation. J. Neurosci. Res. 55: 17–23, 1999. © 1999 Wiley‐Liss, Inc.     

Characterisation of microglia isolated from adult human and rat brain

A method has been developed to isolate microglia from adult human and rat brain cell suspensions by rosette formation via Fc receptors. Immunocytochemical characterisation of the cells immediately following isolation and after 7-10 days in vitro with a panel of monoclonal antibodies has demonstrated that microglia from adult brain have the phenotypic characteristics and phagocytic capacity of mononuclear phagocytes, but lack the hydrolytic enzyme, non-specific esterase. The ability to isolate rapidly a purified population of microglia from adult brain provides a means for investigating mechanisms of activation and differentiation of tissue macrophages, which could elucidate their role in inflammation of the central nervous system.     

Characterization of a novel brain-derived microglial cell line isolated from neonatal rat brain.

We observed highly aggressively proliferating immortalized (HAPI) cells growing in cultures that had been enriched for microglia. The cells were initially obtained from mixed glial cultures prepared from 3-day-old rat brains. HAPI cells are typically round with few or no processes when cultured in 10% serum containing medium. As the percentage of serum in the medium is decreased, the HAPI cells have more processes. HAPI cells stain for the isolectin B4, OX-42, and GLUT5, which are markers for microglial cells, but the cells do not immunolabel with A2B5, a marker of cells in the oligodendroglial cell lineage, or with the astrocyte-specific marker, glial fibrillary aciidic protein (GFAP). In addition, HAPI cells are capable of phagocytosis. We conclude that HAPI cells are of microglia/macrophage lineage. Exposing HAPI cells to lipopolysaccharide (LPS) induces the mRNAs for tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS). LPS exposure also induces secretion of TNF-alpha and production of nitric oxide (NO) in HAPI cells. Because activation of microglia is associated with an increase in iron accumulation and ferritin expression, we tested the hypothesis that iron status affects the production of TNF-alpha and NO. Our studies demonstrate that both iron chelation and iron loading diminished the LPS-induced effect of TNF-alpha and NO. The results of this study indicate that HAPI cells possess the characteristics of microglia/brain macrophages, providing an alternative cell culture model for the study of microglia. In addition, we demonstrate that the activation of microglial cells could be modified by iron.     

Cytokine modulation of murine microglial cell superoxide production

Activated microglia may contribute to two opposite effects during inflammation within the central nervous system: host defense against microorganisms and neuronal injury. Each of these processes may be mediated by the generation of reactive oxygen intermediates by activated microglia. We investigated the effects of two proinflammatory cytokines, interferon (IFN)-β and tumor necrosis factor (TNF)-α, and of the anti-inflammatory cytokine, transforming growth factor (TGF)-β, on murine microglial cell superoxide (O₂) production upon stimulation with phorbol myristate acetate (PMA). Priming of microglia with IFN-β or TNF-α resulted in a dose-dependent enhancement of O₂ release in response to PMA. The priming effects of these two cytokines were additive, suggesting that they acted by independent mechanisms. We also found that IFN-β and TNF-β stimulated the release of bioactive TGF-β and that treatment of microglial cell cultures with TGF-β antagonized the priming effects of IFN-β and TNF-α on O₂ production. The results of this study have implications for understanding the mechanisms by which cytokines and microglia may contribute to host defense as well as to injury of the brain. © 1995 Wiley-Liss, Inc.     

Dynamic motility of microglia: purinergic modulation of microglial movement in the normal and pathological brain

Microglia have highly branched and motile cell processes and constantly screen the brain parenchyma under physiological conditions. In response to pathological stimuli, microglia exhibit morphological changes and migrate toward the lesioned site, where they play important roles in inflammatory reactions and neuronal damage. Within minutes of brain damage, microglial processes rapidly extend toward the injured site. The chemoattractive response is triggered by ATP released at the site of injury and the consequent activation of the purinergic receptor P2Y₁₂R on microglia. In addition to the purinergic signals, various neuronal signaling molecules actively and negatively control microglial motility, which is important for regulating the functional activation of microglia in response to pathology. In this review, we focus on the dynamic motion of microglia and describe several key molecules regulating microglial motility in normal and pathological brain tissues. © 2011 Wiley‐Liss, Inc.     

Lipocortin 1 immunoreactivity identifies microglia in adult rat brain

Immunohistochemical localization of two Ca⁺⁺-binding proteins, Lipocortin 1 (LC1) and S100-β, demonstrates two distinct classes of primitive glia in the floor plate of rat embryos. With proper fixation (formalin-lysine-periodate-acetic acid), dendritic glia in the CNS of adult rats also apparently stain for either LC1 or S100-β in the ratio of 1:3. In order to further distinguish and identify these two glial classes, we have examined their population density, topography, and responses to localized neuron death. Neurons of the ipsilateral thalamus undergo apoptosis following cortical ablation; the contralateral thalamus serves as control. By eight days post-lesion, the number of LC1 cells in the ipsilateral thalamus has increased >4-fold, the increase comprising primarily activated phagocytes adjacent to degenerating neurons. The S100-β glia in the same region are virtual- ly indistinguishable from control; but background staining (apparently representing extracellular S100-β) is increased. Thus, the responses of dendritic LC1 glia resemble those previously described for microglia and are quite different from the astrocytes identified by S100-β immunoreactivity. Both dendritic and activated forms of LC1 glia stain with the microglial marker, Griffonia simplicifolia iso-lectin B₄. However, before the correspondence of LC1 glia and microglia can be confirmed, two anomalies require resolution: (1) the LC1 glia are greater in number and more evenly distributed than microglia marked with other methods; (2) the dendritic LC1 glia apparently are progeny of primitive glia that form the midline raphe of the embryonic floor plate. The participation of LC1 glia in the removal of CNS debris supports the hypothesis that LC1 plays anti-inflammatory and/or immunosuppressive roles in phagocytes. © 1993 Wiley-Liss, Inc.     

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.     

Purinergic receptors on microglial cells: functional expression in acute brain slices and modulation of microglial activation in vitro

Microglial cells are the pathologic sensors in the brain. ATP released from damaged cells is a candidate for signalling neural injury to microglia. Moreover, ATP is an extracellular messenger for propagating astrocyte activity in the form of Ca2+ waves. To test for the functional expression of purinoreceptors in microglial cells we employed the patch-clamp technique in acute slices of adult mouse brain. ATP triggered a nonselective cationic and a K+ current. Pharmacological screening with purinergic ligands indicated the presence of P2Y1 and P2Y2/4 receptors linked to the activation of a K+ current and P2X receptors, including P2X7, linked to the activation of a nonselective cationic current. These findings suggest that microglial cells in situ express different purinergic receptors with distinct sensitivity and functional coupling. To test for the involvement of purinoreceptors in microglial activation, we stimulated cultured microglial cells with lipopolysaccharide and measured the release of tumour necrosis factor alpha, interleukin-6, interleukin-12 and macrophage inflammatory protein 1alpha, induction of K+ outward currents and nitric oxide release. All these parameters were reduced in the presence of purinergic ligands, indicating that purinergic receptor activation attenuated indicators of microglial activation.     

Production of hydroxyeicosatetraenoic acids and prostaglandins by a novel rat microglial cell line

We have established a clonal cell line derived from rat microglia that proliferates in response to macrophage-colony stimulating factor (CSF-1). Like primary neonatal microglia, these cells (named RTMGL1) exhibit a ramified morphology, bind isolectin B4, express CD68 and are weakly positive for CD11b and MHC class II. CSF-1-dependent proliferation requires intact signal transduction through several pathways. RTMGL1 synthesize multiple cyclooxygenase (COX) products including 11- and 15-hydroxyeicosatetraenoic acid (HETE) and express COX-2. RTMGL1 synthesize 5-HETE from arachidonic acid (AA) likely via a 5-lipoxygenase (LO). Thus, RTMGL1 have morphological and histological characteristics of primary microglia and metabolize AA via both COX and LO pathways.     

NaV1.5 sodium channels in a human microglial cell line

Microglial cells are the major immuno-competent cells in the mammalian brain where they play a crucial role in maintaining the CNS environment in the face of various potentially pathological insults. We have used electrophysiological and pharmacological methods to study a microglial cell line (C13-NJ) derived from the human CNS. In whole-cell patch clamp experiments we identified an inward current that exhibited biophysical hallmarks of a classical voltage-gated Na⁺ channel. This identification was confirmed by further experiments in which the current was eliminated by removal of Na⁺ from the bathing medium. Relatively weak inhibition by TTX (30 ± 3% at 500 nM) and sensitivity to 100 μM Zn²⁺ suggested that this current was predominantly mediated by the cardiac sodium channel isoform Na_V1.5. Sodium current density was not altered by treatment with either lipopolysaccharide or beta-amyloid 1–42. The presence of the Na_V1.5 subunit in microglial cells is discussed with respect to its reported roles in phagocytosis, proliferation and migration of other non-cardiac cells.     

Chemokine modulation of high-conductance Ca(2+)-sensitive K(+) currents in microglia from human hippocampi

During acute pathological processes, microglia transform into an activated state characterized by a defined morphology and current profile, and are recruited to injury sites by chemokines. No information is available on the ion channels and the mode of action of chemokines in microglia in brain slices from humans with a chronic pathology. Thus, patch-clamp recordings of microglia were performed in hippocampal slices from seven patients who underwent surgery for pharmaco-resistant epilepsy. Cells were identified as microglia by positive labelling with fluorescein-conjugated tomato lectin before recording. All the recorded cells had an ameboid morphology characteristic of activated microglia. However, they had a high input resistance (3.6 G omega), a zero-current resting potential of -16 mV, and lacked Na+ currents, inwardly rectifying and delayed rectifying K+ currents such as non-activated microglia. Importantly, recorded cells expressed Ca2+-sensitive outward currents that activated at 0 mV with non-buffered intracellular Ca2+ and were sensitive to 1 mm tetraethylammonium (TEA). The estimated single-channel conductances were 187 pS in cell-attached and 149 pS in outside-out patches, similar to those of high-conductance Ca2+-dependent K+ channels. The chemokine MIP1-alpha increased whole-cell outward current amplitudes measured at +60 mV by a factor of 3.3. Thus, microglia in hippocampi from epileptic patients express high-conductance Ca2+-dependent K+ channels that are modulated by the chemokine MIP1-alpha. This modulation may contribute to the migratory effect of MIP1-alpha on microglia.     

Tracing of fluoro-gold prelabeled microglia injected into the adult rat brain

In light of a recent interest in the transplantation of cultured microglial cells, we have examined the use of the fluorescent dye Fluoro-Gold (FG) as a tracer for these cells. Following injection into the adult rat brain, FG prelabeled microglial cells were readily traceable for up to 2 weeks with minimal labeling of endogenous cell populations. Some of the injected cells differentiated into ramified microglial cells as a result of exposure to the adult CNS environment. Injection of free FG into the adult rat brain resulted in the widespread labeling of neurons and perivascular cells, but not endogenous microglial cells, indicating that perivascular cells, but not resting microglia, are actively pinocytotic cells of the CNS. Our results show that FG is an effective label for the tracing of transplanted microglial cells. GLIA 23:84–88, 1998. © 1998 Wiley-Liss, Inc.     

In vitro model of microglial deramification: ramified microglia transform into amoeboid phagocytes following addition of brain cell membranes to microglia-astrocyte cocultures

Changes in the morphology of ramified microglia are a common feature in brain pathology and culminate in the appearance of small, rounded, microglia-derived phagocytes in the presence of neural debris. Here, we explored the effect of adding brain cell membranes on the morphology of alphaMbeta2-integrin (CD11b/CD18, CR3) positive microglia cultured on a confluent astrocyte substrate as an in vitro model of deramification. Addition of brain membranes led to a loss of microglial ramification, with full transformation to small, rounded, macrophages at 20-40 microg/ml. Time course studies showed a rapid response, with first effects at 1-3 hours, and full transformation at 24-48 hours. Removal of cell membranes and exchange of the culture medium led to a similarly rapid process of reramification. Comparison of cell membranes from different tissues at 20 microg/ml showed strong transforming effect for the brain, more moderate for kidney and liver, and very weak for spleen and skeletal muscle. Fluorescent labeling of brain membranes revealed uptake by almost all rounded macrophages, by a subpopulation of glial fibrillary acidic protein (GFAP)-positive astrocytes, but not by ramified microglia. Phagocytosis of inert fluorobeads did not lead to a transformation into macrophages but their phagocytosis was inhibited by brain membranes, pointing to a saturable uptake mechanism. In summary, addition of brain cell membranes and their phagocytosis leads to a rapid and reversible loss of ramification. The differences in transforming activity from different tissues and the absence of effect from phagocytosed fluorobeads suggest, however, the need for a second stimulus following the phagocytosis of cell debris.     

Progressive activation of adult microglial cells in vitro

Bulk-isolated microglial cells from the adult rat brain grown in N2 medium supplemented with 10% fetal calf serum survived for at least 2 weeks, and their purity was > 99% at day 1 and > 93% at day 7. The phenotype of freshly plated cells was comparable to that of “resting,” ramified microglia in vivo. With time in culture and with different schedules, depending on the parameter considered, microglia acquired antigenic (e.g., positivity for vimentin, ED1, major histocompatibility complex class I antigens, leukocyte common antigen, and to a lesser extent CD4) and functional (e.g., proliferation, phagocytosis) features characteristic of “activated” microglia as described in situ. Production of nitrite and prostaglandin E₂ in response to lipopolysaccharide increased greatly with time in culture. Phagocytosis was also accompanied by increased release of nitrite and prostaglandin E₂, the latter being more affected than the first by the age of the cultures. The culture system described may be suitable to study the factors that can modulate “activation” of adult microglia. © 1996 Wiley-Liss, Inc.     

Reactive cell proliferation and microglia following injury to the rat brain

The non–astrocytic cells which proliferate in the rat brain after the induction of an area of necrosis have been characterized and counted by means of combined in vivo bromodeoxyuridine (BrdU) administration and immuno–histochemical demonstration of glial fibrillary acid protein (GFAP), vimentin, Ricinus communis agglutinin 120 (RCA–1), Griffonia simplicifolia B4 isolectin (GSI–B4), keratan sulphate (KS), carbonic anhydrase C (CA.C), transferrin (TF) and ferritin. Two days after the injury, 7.5% of the proliferating cells were GFAP–positive reactive astrocytes, 5.7% were RCA–1–positive cells and 17.4% were GSI–B4–positive cells. Lectin–binding cells had the microscopic and ultrastructural aspects of microglia; they proliferated around the needle track and in the corpus callosum. Microglia represented a large fraction of the proliferating cells. Evidence is presented for the origin of at least a proportion of perilesional astrocytes and microglia from the periventricular matrix, and of microglia from blood precursors. Other non–proliferating microglia cells transiently appeared in the normal brain around the wound, in agreement with the existence of two different microglia cell populations reacting with different modalities to an area of necrosis.     

Serum matrix metalloproteinase levels correlate with brain injury in human immunodeficiency virus infection

Circulating levels of specific matrix metalloproteinases (MMPs; 1 and 7) were evaluated as correlates of brain injury in eight individuals in advanced human immunodeficiency virus (HIV) infection. Neurological status was quantified in vivo with automated segmentation algorithms and with diffusion tensor imaging. Both metalloproteinases correlated with microstructural brain alterations and the degree of atrophy. MMPs may influence neurological outcome through involvement in neuroimmune response, blood-brain barrier permeability, leukocyte migration, and MMP-mediated neurotoxicity.