Phospholipids modulate superoxide and nitric oxide production by lipopolysaccharide and phorbol 12-myristate-13-acetate-activated microglia

Microglial activation and inflammatory processes have been implicated in the pathogenesis of a number of neurodegenerative disorders. Recently, peroxynitrite (ONOO(-)), the reaction product of superoxide (O(2)(-)) and nitric oxide (NO) both of which can be generated by activated microglia, has been demonstrated to act as a major mediator in the neurotoxicity induced by activated microglia. On the other hand, phospholipids such as phosphatidylserine (PS) and phosphatidylcholine (PC) have been reported to modulate the immune function of phagocytes. We therefore evaluated the effects of liposomes which comprise both PS and PC (PS/PC liposomes) or PC only (PC liposomes) regarding the production of both O(2)(-) and NO by lipopolysaccharide (LPS)/phorbol 12-myristate-13-acetate (PMA)-activated microglia using electron spin resonance (ESR) spin trap technique with a DEPMPO and Griess reaction, respectively. Pretreatment with PS/PC liposomes or PC liposomes considerably inhibited the signal intensity of O(2)(-) adduct associated with LPS/PMA-activated microglia in a dose-dependent manner. In addition, pretreatment with PS/PC liposomes also significantly reduced LPS/PMA-induced microglial NO production. In contrast, pretreatment with PC liposomes had no effect on the NO production. These results indicate that PS/PC liposomes can inhibit the microglial production of both NO and O(2)(-), and thus presumably prevent a subsequent formation of ONOO(-). Therefore, PS/PC liposomes appear to have both neuroprotective and anti-oxidative properties through the inhibition of microglial activation.     

Chemiluminescence of mononuclear cells is enhanced during antigen recognition

Stimulation of phagocytes by several cytokines causes superoxide generation and consequently chemiluminescence. Since antigen-activated lymphocytes generate cytokines, we investigated whether antigen recognition by mononuclear cells, which contain both lymphocytes and monocytes, is accompanied by changes in lucigenin-dependent chemiluminescence. Mononulcear cells which underwent antigen-induced proliferation showed a delayed rise in lucigenin-dependent chemiluminescence in the absence of other stimuli. The common recall antigen Candida albicans increased spontaneous chemiluminescence of mononuclear cells from unselected donors up to 20-fold over control values after 48–72h of culture. With Rabies virus vaccine as specific antigenic stimulus, only mononuclear cells from rabies immunized individuals responded with enhanced delayed chemiluminescence. In contrast to opsonized zymosan and phorbol myristate acetate, antigens induced no oxidative burst within one hour after addition. Delayed mononuclear cel chemiluminescence was inhibited by the superoxide scavenger superoxide dismutase and by di-phenylene iodonium, a selective inhibitor of the phagocyte NADPH oxidase. A neutralizing monoclonal antibody against interferon-gamma completely abrogated antigen-induced chemiluminescence. Recombinant interferon-gamma by itself induced delayed mononuclear cell chemiluminescence. Thus, antigen-induced delayed mononuclear cell chemiluminescence represents activation of phagocyte NADPH oxidase by interferon-gamma generated by activated lymphocytes.     

Activation of BV2 microglia by lipopolysaccharide triggers an inflammatory reaction in PC12 cell apoptosis through a toll-like receptor 4-dependent pathway

Microglia play an important role in neuronal protection and damage. However, the molecular and cellular relationship between microglia and neurons is unclear. We carried out a prospective study to detect that activation of BV2 microglia induced PC12 cell apoptosis in vitro through the TLR4/adapter protein myeloid differentiation factor 88 (MyD88)/nuclear factor-κB (NF-κB) signaling pathway. BV2 microglia were treated with different concentrations of LPS for 24 h. Western blot was utilized to detect the expression of TLR4 and the downstream signaling pathway. The level of inflammatory mediator was quantified using a specific ELISA kit. The supernatant of 10 μg/ml LPS-treated BV2 cells was used as conditioned medium (CM). PC12 cells were co-culture with CM for 24 h. Cell viability was determined by MTT assay and cell apoptosis was tested by flow cytometry. BV2 microglia were treated with 10, 20, or 30 μg/ml LPS for 24 h. The expression of TLR4, MyD88, and NF-κB significantly increased. When PC12 cells were co-cultured with CM for 24 h, cell viability decreased. CM up-regulated the Bax level and down-regulated the Bcl-2 protein level in PC12 cells. PC12 cells pretreated with interleukin-1 receptor antagonist (IL-1RA) for 30 min, significantly alleviated CM-induced PC12 cell apoptosis. These results suggest that BV2 microglia activated by LPS triggered TLR4/MyD88/NF-κB signaling pathway that induced the release of IL-1β and could participate in the PC12 cells injury.     

Cu/Zn- and Mn-superoxide dismutases are specifically up-regulated in neurons after focal brain injury

In previous studies, we have demonstrated that damaged neurons within a boundary area around necrosis fall into delayed cell death due to the cytotoxic effect of microglial nitric oxide (NO), and are finally eliminated by activated microglia. In contrast, neurons in a narrow surrounding region nearby this boundary area remain alive even though they may encounter cytotoxic NO. To investigate the mechanism by which neurons tolerate this oxidative stress, we examined the in vitro and in vivo expression levels of superoxide dismutase (SOD) under pathological conditions. Results from our in situ hybridization and immunohistochemical studies showed up-regulation of Cu/Zn-SOD only in neurons outside the boundary area, whereas up-regulation of Mn-SOD was detected in both neurons and glial cells in the same region. In vitro experiments using rat PC12 pheochromocytoma and C6 glioma cell lines showed that induction of both Cu/Zn- and Mn-SOD mRNA could only be detected in PC12 cells after treatment with NO donors, while a slight induction of Mn-SOD mRNA alone could be seen in C6 glioma cells. The mechanism of resistance toward oxidative stress therefore appears to be quite different between neuronal and glial cells. It is assumed that these two types of SOD might play a critical role in protecting neurons from NO cytotoxicity in vivo, and the inability of SOD induction in damaged neurons seems to cause their selective elimination after focal brain injury.     

Paraquat-Induced Cell Death in PC12 Cells

Paraquat was taken up by PC12 cells in a carrier-mediated, saturable manner. When PC12 cells were permeabilized with digitonin (50 g/ml) lipid peroxidation was observed after paraquat treatment in the presence of NADPH and chelated iron. The fact that lipid peroxidation preceded the appearance of LDH release provides positive evidence that lipid peroxidation may be one of the important factors leading to cytotoxicity of cells. Furthermore, the fact that addition of superoxide dismutase, catalase and promethazine efficiently blocked the malondialdehyde formation and attenuated the cell death indicated the involvement of reactive oxygen radicals in mediating the cytotoxicity induced by paraquat. Taken together the results present in vitro evidence that neurotoxicity of paraquat may be a consequence of cellular lipid peroxidation, which leads to cell death and may have great implications in assessing the risk of exposure to paraquat in Parkinson's disease.     

Amyloid precursor protein-processing products affect mononuclear phagocyte activation: pathways for sAPP- and Abeta-mediated neurotoxicity

Increasing evidence strongly supports the role of glial immunity in the pathogenesis of Alzheimer's disease (AD). To investigate such events we have developed cell systems mimicking the interactions between beta-amyloid precursor protein (APP)-expressing neurons and brain mononuclear phagocytes (MP; macrophages and microglia). MP were co-cultured with neuronal cells expressing wild type APP or familial AD-linked APP mutants. The latter was derived from recombinant adenoviral constructs. Neuronal APP processing products induced MP activation, reactive oxygen species, and neurotoxic activities. These occurred without the addition of pro-inflammatory cytokines and were reversed by depletion of amyloid beta-peptide (Abeta) and secreted APP (sAPP). Neurotoxic activities were diminished by superoxide dismutase mimetics and NMDA receptor inhibitors. Microglial glutamate secretion was suppressed by the cystine-glutamate antiporter inhibitor and its levels paralleled the depletion of sAPP and Abeta from conditioned media prepared from APP-expressing neurons. The excitotoxins from activated MP were potent enough to evoke recombinant NMDA receptor-mediated inward currents expressed in vitro in the Xenopus oocytes. These results demonstrate that neuronal APP-processing products can induce oxidative neurotoxicity through microglial activation.     

Effects of the pig renal epithelial cell line LLC-PK1 and its conditioned medium on the phenotype of porcine microglia in vitro

Microglia are dispersed throughout the central nervous system. Under physiological circumstances they display a 'ramified' resting phenotype. In different neuropathologies microglia reversibly transform into the activated form, an amoeboid phagocyte with a broad spectrum of immune effector functions. In this study, a coculture of porcine microglia and the pig renal epithelial cell line LLC-PK1 was used to investigate microglial cell biology. The morphology of the cocultures was elucidated as well as the functionality of the microglia cells by proliferation, superoxide and phagocytosis assays. Our results demonstrate that direct intercellular contact between the two cell types was necessary for microglia to acquire a ramified morphology. Moreover, the conditioned medium of the renal cells promoted proliferation of microglia, inhibited giant cell formation and stimulated microglia to retain their capability to generate superoxide and to perform phagocytosis. In conclusion, we have constructed a cell culture system showing differentiation of microglia in vitro and keeping them in optimal conditions.     

Effects of S‐adenosylhomocysteine and homocysteine on DNA damage and cell cytotoxicity in murine hepatic and microglia cell lines

Limited research has been performed on S‐adenosylhomocysteine (SAH) or homocysteine (Hcy)‐evoked cell damage in hepatic and neuronal cells. In this study, we assessed effects of SAH or Hcy on cell cytotoxicity and DNA damage in hepatic and neuronal cells and attempted to find the underlying mechanism. Cell cytotoxicity and DNA damage were evaluated in murine hepatic cells (BNL CL.2 cell line) and microglia cells (BV‐2 cell line) with SAH or Hcy treatment for 48 h. The influences of SAH or Hcy on lipid peroxidation and DNA methylation were also measured in both cell lines. SAH (5–20 μM) or Hcy (1–5 mM) dose dependently inhibited cell cytotoxicity and enhanced DNA damage in both types of cells. Furthermore, SAH treatment markedly increased intracellular SAH levels and DNA hypomethylation, whereas Hcy caused minimal effects on these two parameters at much higher concentrations. Hcy significantly induced lipid peroxidation, but not SAH. The present results show that SAH might cause cellular DNA damage in hepatic and microglia cells by DNA hypomethylation, resulting in irreversible DNA damage and increased cell cytotoxicity. In addition, higher Hcy could induce cellular DNA damage through increased lipid peroxidation and DNA hypomethylation. We suggest that SAH is a better marker of cell damage than Hcy in hepatic and microglia cells. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:349–356, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20298     

Signaling of ATP receptors in glia-neuron interaction and pain

ATP causes the activation of p38 or ERK1/2, mitogen activated protein kinases (MAPKs) resulting in the release of tumor necrosis factor-alpha (TNF) and Interleukin-6 (IL-6) from microglia. We examined the effect of TNF and IL-6 on the protection from PC12 cell death by serum deprivation. When PC12 cells were incubated with serum-free medium for 32 hr, their viability decreased to 30 %. IL-6 alone slightly protected the death of PC12 cells, whereas TNF alone did not show any protective effect. In the meanwhile, when PC12 cells were pretreated with TNF for 6 hr and then incubated with IL-6 under the condition of serum-free, the viability of PC12 cells dramatically increased. TNF induced an increase of IL-6 receptor (IL-6R) expression in PC12 cells at 4-6 hr. These data suggested that 6 hr pretreatment with TNF increased IL-6R expression in PC12 cells, leading to an enhancement of IL-6-induced neuroprotective action.To elucidate the role of p38 in pathological pain, we investigated whether p38 is activated in the spinal cord of the neuropathic pain model. In the rats displaying a marked allodynia, the level of phospho-p38 was increased in the microglia of injury side in the dorsal horn. Intraspinal administration of p38 inhibitor suppressed the allodynia. These results demonstrate that neuropathic pain hypersensitivity depends upon the activation of p38 signaling pathway in microglia in the dorsal horn following peripheral nerve injury.     

Phycocyanin protects INS-1E pancreatic beta cells against human islet amyloid polypeptide-induced apoptosis through attenuating oxidative stress and modulating JNK and p38 mitogen-activated protein kinase pathways

It is widely accepted that human islet amyloid polypeptide (hIAPP) aggregation plays an important role in the loss of insulin-producing pancreatic beta cells. hIAPP-induced cytotoxicity is mediated by generation of reactive oxygen species (ROS). Phycocyanin (PC) is a natural compound from blue-green algae that is widely used as food supplement. Currently, little is known about the effects of PC on beta cells with the presence of hIAPP. The aim of this study was to investigate the in vitro protective effects of PC on INS-1E rat insulinoma beta cells against hIAPP-induced cell death, as well as the underlying mechanisms. Our results showed that hIAPP-induced cell death with apoptotic characteristics including growth inhibition, chromatin condensation and DNA fragmentation. However, cytotoxicity of hIAPP was significantly attenuated by co-incubation of the cells with PC. The results of Western blotting showed that activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP) in hIAPP-treated cells was blocked by PC. Moreover, PC significantly prevented the hIAPP-induced overproduction of intracellular ROS and malondialdehyde (MDA), as well as changes in activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) enzymes. Furthermore, hIAPP triggered the activation of mitogen-activated protein kinases (MAPKs), and these effects were effectively suppressed by PC. Taken together, our results suggest that PC protects INS-1E pancreatic beta cells against hIAPP-induced apoptotic cell death through attenuating oxidative stress and modulating c-Jun N-terminal kinase (JNK) and p38 pathways.     

Neutrophil stimulation and priming by direct contact with activated human T lymphocytes.

The concept that T lymphocytes regulate neutrophil function has an important implication in the understanding of the role of these cells in immunity against infection and in inflammatory diseases, but evidence for this concept is primarily derived from the effects of lymphokines on neutrophils. We now present evidence to show that living or paraformaldehyde-fixed mitogen-activated T lymphocytes, as well as an activated T cell line (HUT-78), induce by cell-cell contact, an oxygen-dependent respiratory burst measured by both the lucigenin-dependent chemiluminescence assay and superoxide production. Neutrophils reacted with purified human T lymphocytes which had been activated by culture in the presence of PHA and PMA for 72 h showed a marked and significant respiratory burst compared with neutrophils treated with T lymphocytes cultured in the absence of these mitogens. Similar results were observed with the paraformaldehyde-fixed T cell line (HUT-78). The ability to stimulate neutrophils required intact paraformaldehyde-fixed T cells, and neutrophil stimulation failed to occur if the T cells and neutrophils were separated by membrane filters. mAb to TNF-alpha, and TNF-beta blocked the ability of rTNF-alpha and TNF-beta to stimulate neutrophils but did not block the neutrophil response induced by activated T cells. Pretreatment of neutrophils with the activated T lymphocytes enhanced the response to the tripeptide, FMLP. It is therefore conceivable that activated T lymphocytes attracted at sites of inflammation influence neutrophil activity by direct plasma membrane interaction which clearly represents an efficient microbial defence mechanism, minimizing tissue damage during inflammation.     

Microglia enhance beta-amyloid peptide-induced toxicity in cortical and mesencephalic neurons by producing reactive oxygen species

The purpose of this study was to assess and compare the toxicity of beta-amyloid (Abeta) on primary cortical and mesencephalic neurons cultured with and without microglia in order to determine the mechanism underlying microglia-mediated Abeta-induced neurotoxicity. Incubation of cortical or mesencephalic neuron-enriched and mixed neuron-glia cultures with Abeta(1-42) over the concentration range 0.1-6.0 microm caused concentration-dependent neurotoxicity. High concentrations of Abeta (6.0 microm for cortex and 1.5-2.0 microm for mesencephalon) directly injured neurons in neuron-enriched cultures. In contrast, lower concentrations of Abeta (1.0-3.0 microm for cortex and 0.25-1.0 microm for mesencephalon) caused significant neurotoxicity in mixed neuron-glia cultures, but not in neuron- enriched cultures. Several lines of evidence indicated that microglia mediated the potentiated neurotoxicity of Abeta, including the observations that low concentrations of Abeta activated microglia morphologically in neuron-glia cultures and that addition of microglia to cortical neuron-glia cultures enhanced Abeta-induced neurotoxicity. To search for the mechanism underlying the microglia-mediated effects, several proinflammatory factors were examined in neuron-glia cultures. Low doses of Abeta significantly increased the production of superoxide anions, but not of tumor necrosis factor-alpha, interleukin-1beta or nitric oxide. Catalase and superoxide dismutase significantly protected neurons from Abeta toxicity in the presence of microglia. Inhibition of NADPH oxidase activity by diphenyleneiodonium also prevented Abeta-induced neurotoxicity in neuron-glia mixed cultures. The role of NADPH oxidase-generated superoxide in mediating Abeta-induced neurotoxicity was further substantiated by a study which showed that Abeta caused less of a decrease in dopamine uptake in mesencephalic neuron-glia cultures from NADPH oxidase-deficient mutant mice than in that from wild-type controls. This study demonstrates that one of the mechanisms by which microglia can enhance the neurotoxicity of Abeta is via the production of reactive oxygen species.     

Cytochrome P450 and steroid hydroxylase activity in mouse olfactory and vomeronasal mucosa

Neopterin and 7,8-dihydroneopterin, two compounds which are secreted by activated macrophages, have been shown to interfere with radicals generated by cellular and certain chemical systems. Reduced pterins were reported to scavenge whereas aromatic pterins promoted or reduced radical mediated reactions or had no effect. However, recently it was found that high concentrations of 7, 8-dihydroneopterin enhanced luminol dependent chemiluminescence and T-cell apoptosis, suggesting an enhancement of free radical formation. In this study hydroxylation of salicylic acid was used for detection of hydroxyl radicals. It is shown that in solutions of 7,8-dihydroneopterin hydroxyl radicals were formed in the absence of any radical source. The presence of EDTA chelated iron enhanced hydroxyl radical formation. Whereas the addition of iron accelerated the hydroxylation reaction, 7,8-dihydroneopterin was responsible for the amount of hydroxylation products. In the presence of superoxide dismutase or catalase, as well as by helium purging, hydroxylation was inhibited. Our data suggest that in solutions of 7, 8-dihydroneopterin superoxide radicals are generated which are converted to hydroxyl radicals by Fenton or Haber-Weiss type reactions. While superoxide might be generated during autoxidation of ferrous iron, dihydroneopterin seems to be involved in regeneration of ferrous iron from the ferric form.     

Regulation of the growth and differentiation of a human monocytic cell line by lymphokines. I. Induction of superoxide anion production and chemiluminescence

The U937 human monocytic cell line was studied to determine its ability to generate a respiratory burst after stimulation with phorbol myristate acetate (PMA) or opsonized zymosan. U937 cells cultured in normal medium produced virtually no superoxide anion or chemiluminescence in response to either stimulus. In contrast, U937 cells cultured in medium containing soluble factors from activated lymphocytes produced significant O2- and chemiluminescence when stimulated with PMA or opsonized zymosan. The chemiluminescence in response to PMA was maximal in U937 cells precultured with these soluble factors for 3 days, whereas maximal responsiveness to opsonized zymosan was not observed until 5 to 6 days of lymphokine exposure. Although this ability to generate a respiratory burst persisted for a number of days in U937 cells that were subsequently recultured in normal medium, this responsiveness was gradually lost in the continued absence of these factors. The data indicate that the U937 monocytic cell line can be activated or induced to differentiate by soluble factors released by activated lymphocytes. In the process, these cells acquire the ability to generate a respiratory burst. The U937 cell line may serve as a useful model for the study of the ontogeny and regulation of the respiratory burst during human monocytic differentiation.     

Macrophage activation for tumor cytotoxicity: induction of tumoricidal macrophages by supernatants of PPD-stimulated Bacillus Calmette-Guérin-immune spleen cell cultures.

Resident peritoneal macrophages from normal mice were activated for tumor cytotoxicity in vitro by co-cultivation with BCG1-immune spleen cells and PPD and by incubation with supernatants of PPD-stimulated BCG-immune spleen cell cultures (lymphokine supernatants). Lymphokine activation of macrophages occurred in unfractionated PC suspensions as well as in macrophage monolayers depleted of nonadherent PC. Tumor cytotoxicity by lymphokine-activated macrophages was evident by 3 to 4 hr of culture in active supernatants, reached maximal levels by 8 to 12 hr. and was absent by 20 hr. Continued incubation in lymphokines or even re-exposure after washing did not maintain macrophage cytotoxicity. The capacity of normal resident macrophages to be activated by lymphokines in vitro progressively decreased and was absent by 20 hr in culture. This decrease did not necessarily reflect cell death; macrophage viability as estimated by exclusion of trypan blue or by phagocytic responses did not change over the 20-hr culture period. The short lived nature of both macrophage tumoricidal capacity and capacity of precursor cells to be activated by lymphokines may function as negative feedback mechanisms in immune reactions.     

A dual role of lipocalin 2 in the apoptosis and deramification of activated microglia

Activated microglia are thought to undergo apoptosis as a self-regulatory mechanism. To better understand molecular mechanisms of the microglial apoptosis, apoptosis-resistant variants of microglial cells were selected and characterized. The expression of lipocalin 2 (lcn2) was significantly down-regulated in the microglial cells that were resistant to NO-induced apoptosis. lcn2 expression was increased by inflammatory stimuli in microglia. The stable expression of lcn2 as well as the addition of rLCN2 protein augmented the sensitivity of microglia to the NO-induced apoptosis, while knockdown of lcn2 expression using short hairpin RNA attenuated the cell death. Microglial cells with increased lcn2 expression were more sensitive to other cytotoxic agents as well. Thus, inflammatory activation of microglia may lead to up-regulation of lcn2 expression, which sensitizes microglia to the self-regulatory apoptosis. Additionally, the stable expression of lcn2 in BV-2 microglia cells induced a morphological change of the cells into the round shape with a loss of processes. Treatment of primary microglia cultures with the rLCN2 protein also induced the deramification of microglia. The deramification of microglia was closely related with the apoptosis-prone phenotype, because other deramification-inducing agents such as cAMP-elevating agent forskolin, ATP, and calcium ionophore also rendered microglia more sensitive to cell death. Taken together, our results suggest that activated microglia may secrete LCN2 protein, which act in an autocrine manner to sensitize microglia to the self-regulatory apoptosis and to endow microglia with an amoeboid form, a canonical morphology of activated microglia in vivo.     

Chemiluminescence,suppression and cytotoxicity in peripheral blood mononuclear cells from solid tumor cancer patients

Summary Chemiluminescence, indomethacin-sensitive suppression, and adherent cell cytotoxicity were measured in peripheral blood mononuclear cells (PBMC) from normal subjects and solid tumor cancer patients. These functions were found to be differentially affected by malignant disease. In cancer patients with disseminated disease, indomethacin-sensitive suppression and chemiluminescence emission were increased to a level significantly higher than normal without a concurrent increase in adherent cell cytotoxic function. In cancer patients with at most minimum residual diseases, the levels of chemiluminescence, indomethacin-sensitive suppression, and adherent cell cytotoxicity found were comparable to those of the normal study population. In vitro stimulation of cells from patients with disseminated disease by phorbol myristic acetate (PMA) increased chemiluminescence overcame the suppressive effects of indomethacin-sensitive suppressor cells, and increased adherent cell cytotoxicity; in cells from patients with at most minimum residual disease, PMA increased chemiluminescence and cytotoxicity without influencing the activity of indomethacin-sensitive suppressor cells. Vaccination of lung cancer patients with Freund's complete adjuvant or Freund's complete adjuvant plus tumor antigen extracts led to increased levels of chemiluminescence and increased levels of adherent cell cytotoxicity without altering indomethacin-sensitive regulatory cell function.     

NADPH oxidase mediates lipopolysaccharide-induced neurotoxicity and proinflammatory gene expression in activated microglia

Parkinson's disease is characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra. We have previously reported that lipopolysaccharide (LPS)-induced degeneration of dopaminergic neurons is mediated by the release of proinflammatory factors from activated microglia. Here, we report the pivotal role of NADPH oxidase in inflammation-mediated neurotoxicity, where the LPS-induced loss of nigral dopaminergic neurons in vivo was significantly less pronounced in NADPH oxidase-deficient (PHOX-/-) mice when compared with control (PHOX+/+) mice. Dopaminergic neurons in primary mensencephalic neuron-glia cultures from PHOX+/+ mice were significantly more sensitive to LPS-induced neurotoxicity in vitro when compared with PHOX-/- mice. Further, PHOX+/+ neuron-glia cultures chemically depleted of microglia failed to show dopaminergic neurotoxicity with the addition of LPS. Neuron-enriched cultures from both PHOX+/+ mice and PHOX-/- mice also failed to show any direct LPS-induced dopaminergic neurotoxicity. However, the addition of PHOX+/+ microglia to neuron-enriched cultures from either strain resulted in reinstatement of LPS-induced dopaminergic neurotoxicity, supporting the role of microglia as the primary source of NADPH oxidase-generated insult and neurotoxicity. Immunostaining for F4/80 in mensencephalic neuron-glia cultures revealed that PHOX-/- microglia failed to show activated morphology at 10 h, suggesting an important role of reactive oxygen species (ROS) generated from NADPH oxidase in the early activation of microglia. LPS also failed to elicit extracellular superoxide and produced low levels of intracellular ROS in microglia-enriched cultures from PHOX-/- mice. Gene expression and release of tumor necrosis factor alpha was much lower in PHOX-/- mice than in control PHOX+/+ mice. Together, these results demonstrate the dual neurotoxic functions of microglial NADPH oxidase: 1) the production of extracellular ROS that is toxic to dopamine neurons and 2) the amplification of proinflammatory gene expression and associated neurotoxicity.     

Involvement of adhesion molecules (CD11a-ICAM-1) in vascular endothelial cell injury elicited by PMA-stimulated neutrophils.

Protective effect of anti-CD11a and anti-ICAM-1 antibodies on the cytotoxicity induced by PMA-stimulated neutrophils was studied using cultured endothelial cells isolated from bovine carotid artery. Anti-CD11a antibody and anti-ICAM-1 antibody inhibited the endothelial cell injury induced by the activated neutrophils in a dose dependent manner. On the other hand, both antibodies themselves had no effect on either the luminol chemiluminescence released out of the activated neutrophils or the adhesion of the neutrophils to the endothelial cell monolayer. These data suggest that these adhesion molecules play some important roles in the vascular endothelial cell injury elicited by activated neutrophils.     

Activated microglia mediate neuronal cell injury via a nitric oxide mechanism.

Activated microglial have been proposed to play a pathogenetic role in immune-mediated neurodegenerative diseases. To test this hypothesis, purified murine neonatal microglial were cocultured with neuronal cells derived from fetal brain. Activation with IFN-gamma and LPS of these cocultures brought about a sharp decrease in uptake of gamma-amino butyric acid and a marked reduction in neuronal cell survival. These effects varied with the density of microglia, the concentrations of the activation signals (IFN-gamma and LPS), and the duration of coculture. Inasmuch as addition of NG-monomethyl-L-arginine blocked these effects, a L-arginine-dependent neurocytotoxic mechanism was implicated. Abundant nitrite, a metabolite of the free radical nitric oxide (NO) derived from L-arginine, was detected in activated microglial/neuronal cell cocultures and in purified microglial cell cultures but not in purified astrocyte or neuronal cell cultures, suggesting that microglial were the principal source of the NO. These findings support the hypothesis that microglia are the source of a neurocytotoxic-free radical, and shed light on an additional mechanism of immune-mediated brain injury.