Activation of murine microglial cell lines by lipopolysaccharide and interferon-gamma causes NO-mediated decreases in mitochondrial and cellular function

Activation of murine microglial and macrophage cell lines with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) resulted in the induction of the inducible form of nitric oxide synthase (NOS) and the release of micromolar amounts of NO into the surrounding medium. The synthesis of NO was associated with increased cellular membrane damage as assessed by trypan blue dye exclusion and the leakage of lactate dehydrogenase into the cell culture medium. However, the synthesis and release of cytokines was largely unaffected. NO-mediated cell damage was also accompanied by a marked decrease in the intracellular levels of reduced glutathione and ATP. In addition, significant inhibition of mitochondrial respiratory chain enzyme activities was seen following cellular activation. However, citrate synthase activity (a mitochondrial matrix enzyme) was not detectable in the extracellular supernatants, suggesting preservation of the integrity of the mitochondrial inner membrane following activation. These effects were largely prevented by the addition of the NOS inhibitor, N-guanidino monomethyl L-arginine during the activation period. Our observations demonstrate that induction of NOS activity in microglia results in damage to the plasma membrane leading to a loss of glutathione, complex-specific inhibition of the mitochondrial electron transport chain and depletion of cellular ATP. Our data suggest that pharmacological modulation of NOS activity in activated microglia in vivo may prevent cellular damage to bystander cells such as neurons, astrocytes and oligodendrocytes, as well as to microglia themselves.     

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.     

Generation and characterization of mouse microglial cell lines

A murine cell line (MMGT1) has been established after transfection of primary microglial cell cultures with a v-myc-containing plasmid. This cell line was comparable with primary microglial cells with respect to morphology, presence of acetylated low density lipoprotein receptor, non-specific estrase, CD63, major histocompatibility complex antigens and CD11, and binding for Ricinus communis agglutinin. Primary microglia as well as MMGT1 cells were negative for glial fibrillary acidic protein. Different MMGT1 strains were obtained after subcloning, two of which resembled histocytes (F4/80 and BM-8). These cell strains, MMGT12 and 16, were able to opsonize latex beads, and could be induced by endotoxins (LPS) to secrete TNF-α, IL-1, IL-6, TGF-β, and EGF. The other subclones had intermediate (MCA519, ER-MP20) or mixed macrophage characteristics and did not react to endotoxin by an increase in TNF-α, IL-1, and TGF-β. Our newly established murine microglia lines may prove to be useful models to study inflammation and repair in the brain.     

Differential expression of carbonic anhydrase isozymes in microglial cell types

Microglial cells have been shown to express carbonic anhydrase. Using carbonic anhydrase histochemistry and immunohistochemistry, different types of cen-tral nervous system microglial cells were detected, which expressed two main carbonic anhydrase (CA) isozymes during the early postnatal stage of development and after peripheral nerve injury in the spinal cord of adult rats. Amoeboid and reactive microglial cells were heavily immunostained for CA-II and CA-III and showed colocalization with complement receptor type 3 and Griffonia Simplicifolia B₄ isolectin. Resting microglial cells in the brain and spinal cord showed faint CA-III staining and were negative for CA-II. These results show that not only CA-II, but also CA-III isozyme is represented in the central nervous system and carbonic anhydrase activity may correlate with metabolic and immunological changes of microglial cells. These data also further strengthen the idea of the mesodermal origin of central nervous system macrophages. © 1993 Wiley-Liss, Inc.     

Generation and characterization of immortalized rat retinal microglial cell lines

Retinal microglia play an important role as resident immunocompetent and phagocytic cells in the event of injury and disease. Retinal microglia and microglia precursor transplantation show a rescue effect in ischemic retina and retinal degeneration. However, studies of retinal microglia have been hampered by the difficulty of obtaining sufficient numbers of microglia. One way to circumvent this difficulty is to establish permanent retinal microglia cell lines. In the present study, we report the generation of immortalized retinal microglia, T‐MG cells, from postnatal day 3 rat retinal tissue using a lentiviral vector encoding SV40 large T antigen. The T‐MG cells exhibited cell‐type‐specific antigens for monocyte/macrophage lineage cells, including CD11b (OX42), ED1 (OX6), and Iba1, and actively phagocytosed latex beads. In addition to primary retinal microglia, T‐MG cells also have the ability to recruit into chemokines. Treatment of T‐MG cells with lipopolysaccharide (LPS) led to increased levels of tumor necrosis factor‐α, interleukin‐1β, and inducible nitric oxide synthase. Genome‐wide microarray analysis showed a less than 1% difference in the genes between the T‐MG cells and the control primary retinal microglia. The T‐MG cells exhibited properties similar to those of the primary retinal microglia and should have considerable utility as an in vitro model for the study of retinal microglia in health and as a curative therapy and an in vivo model for the study of retinal microglia in disease. © 2014 Wiley Periodicals, Inc.     

Localized retinal neuropeptide regulation of macrophage and microglial cell functionality

The functionality of immune cells is manipulated within the ocular microenvironment to protect the sensitive and non-regenerating light-gathering tissue from the collateral damage of inflammation. This is mediated partly by the constitutive presence of immunomodulating neuropeptides. Treating primary resting macrophages with soluble factors produced by the posterior eye induced co-expression of Arginase1 and NOS2. The neuropeptides alpha-melanocyte stimulating hormone and Neuropeptide Y alternatively activated the macrophages to co-express Arginase1 and NOS2 like myeloid suppressor cells. Similar co-expressing cells were found within healthy, but not in wounded retinas. Therefore, the healthy retina regulates macrophage functionality to the benefit of ocular immune privilege.     

Neurotrophins regulate proliferation and survival of two microglial cell lines in vitro

Microglia are thought to play a key role in the development and regeneration of the central nervous system although the mechanisms regulating their presence and activity are not fully understood. Substantial evidence suggests that members of the neurotrophin family such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 and -4 (NT-3/4) have a dramatic effect on both neurons and perineuronal cells. This study employed two murine microglial lines, BV-2 and N9, to examine the action of these neurotrophins on the mitotic activity and survival of microglia in vitro. Neurotrophins were incorporated into the media at the time of plating and cell number and levels of mitochondrial dehydrogenase activity (MTT) were determined at various time points in vitro. NGF increased cell number and MTT levels of both cell lines in a dose-dependent manner. BV-2 was more sensitive to NGF than N9. Similar responses were elicited by BDNF, although the sensitivity of each cell line was different than that found for NGF. NT-3 and NT-4 had no effect on cell proliferation. However, NT-4 had an effect on the survival of BV-2 and N9 cells. The response of these cells to neurotrophins was blocked by K252a, a tyrosine kinase inhibitor, suggesting that actions of neurotrophins were mediated by high-affinity tyrosine kinase receptors (Trk). Immunolocalization studies revealed positive Trk (pan) reactivity in the above cell lines and in primary microglia, but an absence of the low-affinity p75 neurotrophin receptor. Western blot analysis supported the above observations. These studies suggest that in addition to their neurotrophic actions, NGF and BDNF may also regulate microglial dynamics, thereby influencing the surrounding milieu during neuronal regeneration.     

Regulation of microglial cell responses in murine Toxoplasma encephalitis by CD200/CD200 receptor interaction

Under autoimmune inflammatory conditions within the brain, evidence suggests that neurons downregulate microglial activation through CD200/CD200R interaction, which reduces disease severity. To gain insight into the regulation of intracerebral immune reactions by resident brain cells in chronic cerebral infections, the expression of the CD200 antigen and the CD200R as well as the functional role of CD200/CD200R interactions were characterized in murine Toxoplasma encephalitis. In the normal brain of C57BL/6 wild type mice, CD200 was ubiquitously expressed on neurons, their axons, cerebral endothelial cells, and plexus macrophages. CD200R was expressed at very low levels on cerebral macrophages and microglia without differences between CD200^−/− and wild type mice. Infection of C57BL/6 mice with Toxoplasma gondii induced an upregulation of CD200R on microglia and of CD200 on blood vessel endothelial cells. In Toxoplasma encephalitis of CD200^−/− mice, microglial cell numbers strongly increased due to an enhanced proliferation indicated by increased Ki-67 immunoreactivity. In addition, microglial activation was increased in CD200^−/− mice as evidenced by a further upregulation of already high MHC class II levels as well as an increased expression of the anti-parasitic effector molecules, TNF and iNOS. The increased microglial cell activation resulted in a reduced intracerebral parasite burden and an increased survival rate. Thus, in Toxoplasma encephalitis, microglial activity was regulated via CD200/CD200R-mediated interaction further pointing to an intrinsic regulation of brain resident cells under inflammatory CNS conditions.     

Differential regulation of microglial activation by propentofylline via cAMP signaling

A pathological microglial activation is believed to contribute to progressive neuronal damage in neurodegenerative diseases by the release of potentially toxic agents and by triggering reactive astrocytic changes. Using cultured microglia from neonatal rat brains, we investigated the mode of propentofylline action in strengthening cAMP-dependent intracellular signaling. We compared this action with the effects of dibutyryl-cAMP, a cell-permeable cAMP analog. Propentofylline inhibited lipopolysaccharide (LPS)-induced release of both tumor necrosis factor (TNF)-α and interleukin (IL)-1β in a dose-dependent manner within the therapeutic low micromolar range. However, LPS-induced release of IL-6 and NO were not affected by propentofylline. All these differential effects of propentofylline on LPS-induced microglial release were mimicked by the addition of dibutyryl-cAMP. Microglial proliferation and phorbol myristate acetate (PMA)-induced O₂⁻ release were also dose-dependently inhibited by propentofylline as well as dibutyryl-cAMP. These results suggest that propentofylline, probably via reinforcement of cAMP intracellular signaling, alters the profile of the newly adopted immune properties in a way that it inhibits potentially neurotoxic functions while maintaining beneficial functions. This differential regulation of microglial activation may explain the neuroprotective mechanism exerted by propentofylline.     

Characterization of the microglial response in murine scrapie

The nature of the glial and inflammatory cell responses to infection in scrapie–affected brains was studied in terminally–affected mice of Ave scrapie models. There were marked astrocytic and microglial responses. Microglia showed increased staining of the surface antigens F4/80, leucocyte–common antigen, type 3 complement receptor, and elevated endocytotic and lysosomal activity. In all models, the astrocytic and microglial responses were largely restricted to anatomical regions of the brain showing vacuolation and/or plaque formation and pathological accumulations of PrP. Expression of MHC Class II was patchy and present on microglia in the neuropil of areas with the most intense microglial activation and on occasional perivascular macrophages. This microglial response may represent a modified form of inflammatory response.     

Immortalization of murine microglial cells by a v-raf/v-myc carrying retrovirus

A murine cell line (BV-2) has been generated by infecting primary microglial cell cultures with a v-raf/v-myc oncogene carrying retrovirus (J2). BV-2 cells expressed nonspecific esterase activity, phagocytic ability and lacked peroxidase activity. Such cells secreted lysozyme and, following appropriate stimulation, also interleukin 1 and tumor necrosis factor. Furthermore, BV-2 cells exhibited spontaneous anti-Candida activity and acquired tumoricidal activity upon treatment with interferon-gamma. Phenotypically, BV-2 cells resulted positive for MAC1 and MAC2 antigens, and negative for MAC3, glial fibrillary acidic protein (GFAP) and galactocerebroside (GC) antigens. Since BV-2 cells retain most of the morphological, phenotypical and functional properties described for freshly isolated microglial cells, we can conclude that J2 virus infection has resulted in the immortalization of active microglial cells.     

Characterization of peripheral benzodiazepine type sites in a cultured murine BV-2 microglial cell line

It is known that the density of peripheral benzodiazepine receptors (PBR) increases after brain damage. Astrocytes are among the cell types where PBR ligand binding has been detected and may be involved in the response to neuronal injury and regeneration. Consistent with the hypothesis, the apparent density of PBR sites in astrocytes is increased by both cytokines and neurotoxins. However, microglia, the resident macrophages which represent 5–15% of glial cell populations have not been evaluated for the presence of the PBR. In the present study, we report the presence of [³H]Ro5-4864 binding in microglial cells. In particular, we used BV-2 cells, an immortalized cell line of murine microglial cells. High affinity binding of [³H]Ro5-4864 to a single site was detected in membranes prepared from BV-2 cells (K_D = 4.4 nM, B_max = 3,800 fmoles/mg protein). Various ligands for the PBR displaced [³H]Ro5-4864 binding with the following rank order of potencies: PK11195 = Ro5-4864 > FGIN-1-27 > triazolam = diazepam > beta-pro-pyl-beta-carboline-3-carboxylate = clonazepam > lorazepam = flurazepam >> chlordiazepoxide = clorazepate. Subcellular fractionationstudies indicate that the majority of the Ro5-4864 binding sites is in the mitochondrial fraction. The remainder is found in non-mitochondrial cell fractions. The [³H]Ro5-4864 binding observed on intact cells had characteristics similar to those found on membranes. The presence of a high density of PBRs in these cells establish the basis for additional investigations into their possible functional role, if any, in the microglial response to neuronal injury. © 1996 Wiley-Liss, Inc.     

Pepstatin A induces extracellular acidification distinct from aspartic protease inhibition in microglial cell lines

The extrusion of protons is considered a very general parameter of the activation of many kinds of membrane or intracellular molecules, such as receptors, ion channels, and enzymes. We found that pepstatin A caused a reproducible, concentration-related increase in the extracellular acidification rate in two microglial cell lines, Ra2 and 6-3. Washing abolished pepstatin A-induced acidification immediately. However, pepstatin A did not cause the extracellular acidification in other cell types, such as CHO, C6 glioma, and NIH3T3 cells. These observations strongly suggest that pepstatin A interacts with certain membrane proteins specific to both Ra2 and 6-3 cells from outside. N-methylmaleimide and N,N'-dicyclohexylcarbodiimide, inhibitors of H(+)-ATPase, were found to reduce pepstatin A-induced response strongly, while bafilomycin A1, a vacuolar H(+)-ATPase inhibitor, vanadate, a P-type H(+)-ATPase inhibitor, and NaN3, an F1 ATPase inhibitor, virtually did not. 5-(N-ethyl-N-isopropyl) amiloride, an inhibitor of Na(+)/H(+) exchanger isoform 1, greatly enhanced pepstatin-induced response, while amiloride did not. Zn(2+), a voltage-dependent proton channel blocker, did not affect pepstatin-induced response neither. Staurosporine, a nonspecific inhibitor of protein kinase C, inhibited pepstatin A-induced response, while chelerythrine, more selective inhibitor of protein kinase C, greatly enhanced it. H-7 and H-8 did not affected the response. These findings suggest that pepstatin A induces extracellular acidification in microglia cell lines, Ra2 and 6-3, through an N-methylmaleimide- and N,N'-dicyclohexylcarbodiimide-sensitive, but bafilomycin A1-insensitive, ATPase, which seems to be distinct from protein kinase C-dependent process.     

Electrically induced fusion of different human cell types

Summary The fusion of human cells of the cerebrospinal fluid and of the peripheral blood is reported, as well as the fusion of these cells with tomato protoplasts. The cells were fused by applying short-time electric pulses after dielectrophoretic collection. The importance of this method for diagnosis and therapy is discussed and possible applications are mentioned.Dedicated to Professor H. Rennert on the occasion of his 65th birthday     

Differential effector and secretory functions of microglial cell lines derived from BCG-resistant and -susceptible congenic mouse strains

Using congenic strains of mice susceptible (bcg(s)) or resistant (bcg(r)) to BCG, murine microglial cell lines, RR4.R (BCG-resistant) and RR8.S (BCG-susceptible), were established in vitro. Comparative studies revealed that, although phagocytic to a similar extent, RR4.R cells were more active than RR8.S cells in terms of antimycobacterial activity. Interestingly, cells of resistant genotype secreted more nitric oxide, TNF-alpha and IL-12, but less IL-6, than susceptible cells, when stimulated with IFN-gamma alone or in combination with lipopolysaccharide. Nevertheless, no significant differences were observed between the two cell lines in terms of IL-1 beta or IL-10 secretion, or on assessment of cytokine production following exposure to a massive dose of lipopolysaccharide. Overall, these data provide the first evidence that resistant/susceptible genotype influences antimycobacterial activity, NO and cytokine production in microglial cells, the prototype of cerebral macrophages.     

Inducible nitric oxide synthase activity of cloned murine microglial cells

Nitric oxide (NO) is a short-lived diffusable molecule now believed to participate in multiple physiologic functions in the CNS including neurotransmission and the maintenance of vascular tone. Previously, we reported that cell lines obtained by retroviral immortalization of tissue macrophages (M?;) could be induced to synthesize nitrite (NO), a stable end product of the NO synthetic pathway. We have further characterized the induction and activity of this pathway in a panel of seven microglial clones derived from primary embryonic mouse brain cultures. Like M?;, these clones were found to release high levels of NO_-² in response to recombinant interferon-γ (rIFN-γ) as a priming signal together with either bacterial lipopolysaccharide (LPS) or exogenous recombinant tumor necrosis factor-α (rTNF-α). As previously demonstrated for M?;, phagocytosis of zymosan particles during induction of enzyme activity enhanced subsequent NO production, which is of interest in light of the postulated phagocytic role of microglia within the CNS. Biochemical characterization of enzyme activity in intact microglial clones and in isolated cytosolic fractions indicates that the microglial NO synthase present in these murine cell clones represents the M?;-like isotype. These findings suggest that microglial cells could represent a major source of NO within the CNS.     

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.