Yong Beom Lee

Interleukin-15 gene expression in human astrocytes and microglia in culture

INTERLEUKIN-15 (IL-15) is a novel cytokine that has recently been cloned and expressed. IL-15 interacts with components of the IL-2 receptor and exhibits T-cell stimulating activity similar to that of IL-2. In the present study, we investigated the expression of IL-15 in enriched cultures of human fetal astrocytes and microglia using reverse transcription-polymerase chain reaction (RT-PCR) and immunodetection analysis. Low levels of IL-15 were expressed by unstimulated human fetal astrocytes and microglia, and treatment of astrocytes with interleukin-1β (IL-1β), interferon-γ (IFN-γ), or tumor necrosis factor-α (TNF-α) increased the expression of IL-15 at both the mRNA and protein level. Treatment of microglia with IFN-γ and lipopolysaccharide (LPS) similarly increased IL-15 expression in microglia. These findings suggest that IL-15 produced by human fetal astrocytes and microglia may have a role in T cell-mediated immune responses in the human CNS.

Ion channels of human microglia in culture

Macroscopic and microscopic currents have been recorded using human microglia isolated from fetal human brains (12-20 weeks gestation). Within a period of two days following plating of cells, inward K+ currents were small (mean amplitude of 0.3 nA at -100 mV) and outward K+ currents were not observed. For periods in excess of five days after adherence to substrate, an inactivating outward K+ current, sensitive to 4-aminopyridine, was expressed. A slowly rising current, blocked by tetraethylammonium, was also evident in a small population of human microglia. This current was activated with cell depolarization positive to +10 mV and had properties similar to those recently described for a proton current in mouse cells. In early adherent cells (days 1 or 2 after plating), treatment of microglia with interferon-gamma led to the expression of outward K+ current which was lacking in the absence of the treatment. In excised, inside-out patches, two high conductance channels were identified. A calcium-dependent K+ channel (unitary conductance of 106 pS with physiological levels of K+ across the patch) had an open probability of 0.5 with internal Ca2+ at 7 microM and the patch potential at 0 mV. In addition, an anion channel (unitary conductance of 280 pS) was transiently activated with depolarizing or hyperpolarizing steps applied from 0 mV. Characterization of the macroscopic and unitary properties of currents in microglia will have relevance to a description of putative cell functions in the human CNS. In particular, modification of cell electrophysiological properties by various activating stimuli may contribute to signalling processes in CNS pathology.

Free Radical Production in CA1 Neurons Induces MIP-1α Expression, Microglia Recruitment, and Delayed Neuronal Death after Transient Forebrain Ischemia

Several studies report microglial accumulation and activation in the CA1 area in response to transient forebrain ischemia (TFI). Here we examine the possibility that free radicals and chemokines mediate the transient activation of microglia. Free radicals are produced primarily in CA1 pyramidal neurons within 2 h of TFI. Administration of trolox, a vitamin E analog, led to the inhibition of free radical production and recruitment of microglia in the CA1 area. In addition, intrahippocampal injection of Fe2+ triggered free radical production in CA1 neurons, followed by the recruitment and activation of microglial cells into this area. TFI-induced expression of macrophage inflammatory protein-1α (MIP-1α) was increased in CA1 neurons before microglial recruitment, and blocked by trolox. Moreover, the MIP-1α level was upregulated in cultured hippocampal neurons exposed to Fe2+, suggesting an essential role of free radicals in TFI-induced expression of MIP-1α. Intracerebroventricular injection of vMIP-2 (viral macrophage inflammatory protein-2), a broad-spectrum peptide antagonist of chemokine receptors, attenuated microglial recruitment and delayed CA1 neuronal degeneration after TFI. Our data suggest that free radicals produced in CA1 neurons contribute to the recruitment and activation of microglia and neurodegeneration through MIP-1α expression.

Interleukin-10 expression in lipopolysaccharide-activated microglia is mediated by extracellular ATP in an autocrine fashion.

Immune cells have been shown to release ATP into the extracellular space to provide auto- and paracrine purinergic modulation of immune and inflammatory responses. In the present study, we demonstrate that ATP released from lipopolysaccharide (LPS)-stimulated microglia induces interleukin-10 (IL-10) expression in an autocrine manner. The expression as well as secretion of IL-10 by LPS-stimulated microglia was completely inhibited by apyrase, ATP-hydrolyzing enzyme, whereas tumor necrosis factor-&agr; (TNF-&agr;) expression was unaffected. LPS-activated microglia rapidly released a low concentration of ATP (10–20 nM) into the medium, and the nanomolar range of extracellular ATP, ADP, adenosine 5′-O-(3-thiotriphosphate) (ATP-&ggr;-S), and adenosine 5′-O-(2-thiodiphosphate) (ADP-&bgr;-S) induced IL-10 secretion from microglia in a dose-dependent manner. These results suggest that ATP released from LPS-activated microglia and/or a metabolite of ATP (ADP) may induce IL-10 expression through P2Y purinergic receptors.

Interleukin-10 endogenously expressed in microglia prevents lipopolysaccharide-induced neurodegeneration in the rat cerebral cortex in vivo.

A degree of brain inflammation is required for repair of damaged tissue, but excessive inflammation causes neuronal cell death. Here, we observe that IL-10 is expressed in LPS-injected rat cerebral cortex, contributing to neuronal survival. Cells immunopositive for IL-10 were detected as early as 8 h post-injection and persisted for up to 3 d, in parallel with the expression of IL-1β, TNF-α, and iNOS. Double immunofluorescence staining showed that IL-10 expression was localized mainly in activated microglia. Next, we examined the neuroprotective effects of IL-10 using IL-10 neutralizing antibody (IL-10NA). Blockade of IL-10 action caused a significant loss of neurons both 3 d and 7 d after LPS injection. Further, the induction of mRNA species encoding IL-1β, TNF-α, and iNOS, reactive oxygen species (ROS) production, and NADPH oxidase activation, increased after co-injection of LPS and IL-10NA, compared to the levels seen after injection of LPS alone. Taken together, these results clearly suggest that LPS-induced endogenous expression of IL-10 in microglia contributes to neuronal survival by inhibiting brain inflammation.

Effects of ATP and elevated K+ on K+ currents and intracellular Ca2+ in human microglia

We have used whole-cell patch-clamp recordings and calcium microfluorescence measurements to study the effects of ATP and elevated external K+ on properties of human microglia. The application of ATP (at 0.1 mM) led to the activation of a transient inward non-selective cationic current at a cell holding potential of -60 mV and a delayed, transient expression of an outward K+ current activated with depolarizing steps applied from holding level. The ATP response included an increase in inward K+ conductance and a depolarizing shift in reversal potential as determined using a voltage ramp waveform applied from -120 to -50 mV. Fura-2 microspectrofluorescence measurements showed intracellular calcium to be increased following the application of ATP. This response was characterized by an initial transient phase, which persisted in Ca2+-free media and was due to release of Ca2+ from intracellular storage sites. The response had a later plateau phase, consistent with Ca2+ influx. In addition, ATP-induced changes in intracellular Ca2+ exhibited prominent desensitization. Elevated external K+ (at 40 mM) increased inward K+ conductance and shifted the reversal potential in the depolarizing direction, with no effect on outward K+ current or the level of internal Ca2+. The results of these experiments show the differential responses of human microglia to ATP and elevated K+, two putative factors associated with neuronal damage in the central nervous system.

Microglial P2X7 receptor expression is accompanied by neuronal damage in the cerebral cortex of the APPswe/PS1dE9 mouse model of Alzheimer's disease

The possibility that P2X7 receptor (P2X7R) expression in microglia would mediate neuronal damage via reactive oxygen species (ROS) production was examined in the APPswe/PS1dE9 mouse model of Alzheimers disease (AD). P2X7R was predominantly expressed in CD11b-immunopositive microglia from 3 months of age before Aβ plaque formation. In addition, gp91phox, a catalytic subunit of NADPH oxidase, and ethidium fluorescence were detected in P2X7R-positive microglial cells of animals at 6 months of age, indicating that P2X7R-positive microglia could produce ROS. Postsynaptic density 95-positive dendrites showed significant damage in regions positive for P2X7R in the cerebral cortex of 6 month-old mice. Taken together, up-regulation of P2X7R activation and ROS production in microglia are parallel with Aβ increase and correlate with synaptotoxicity in AD.

Iron mediates endothelial cell damage and blood-brain barrier opening in the hippocampus after transient forebrain ischemia in rats

Blood cells are transported into the brain and are thought to participate in neurodegenerative processes following hypoxic ischemic injury. We examined the possibility that transient forebrain ischemia (TFI) causes the blood-brain barrier (BBB) to become permeable to blood cells, possibly via dysfunction and degeneration of endothelial cells in rats. Extravasation of Evans blue and immunoglobulin G (IgG) was observed in the hippocampal CA1-2 areas within 8 h after TFI, and peaked at 48 h. This extravasation was accompanied by loss of tight junction proteins, occludin, and zonula occludens-1, and degeneration of endothelial cells in the CA1-2 areas. Iron overload and mitochondrial free radical production were evident in the microvessel endothelium of the hippocampus before endothelial cell damage occurred. Administration of deferoxamine (DFO), an iron chelator, or Neu2000, an antioxidant, blocked free radical production and endothelial cell degeneration. Our findings suggest that iron overload and iron-mediated free radical production cause loss of tight junction proteins and degeneration of endothelial cells, opening of the BBB after TFI.

Thrombin induces IL-10 production in microglia as a negative feedback regulator of TNF-alpha release.

Interleukin-10 (IL-10), an immunosuppressive cytokine, is produced by monocyte/macrophage lineage cells, T cells, and B cells in the immune system. Here, we show that thrombin induces IL-10 expression in cultured rat microglia. Thrombin treatment increases IL-10 mRNA expression after 3 h and IL-10 release into the culture medium 12 h after thrombin treatment. Neutralizing antibodies against IL-10 significantly enhanced TNF-&agr; release from thrombin-treated microglia. IL-10 release was suppressed by an inhibitor of p38 MAPK, SB203580 but not by an inhibitor of ERK pathway, PD98059, whereas both SB203580 and PD98059 inhibited TNF-&agr; release. These results suggest that thrombin induces IL-10 and TNF-&agr; expression through different signaling mechanisms, and that IL-10 inhibits TNF-&agr; release as a negative feedback regulation.

Cholinergic agonists increase intracellular Ca2+ in cultured human microglia

Microglia are resident phagocytic cells in the central nervous system (CNS), and can be activated in response to various stimuli including neurotransmitters. Using fura-2 imaging, we investigated the effects of carbachol (CCh), a cholinergic agonist, on [Ca2+]i in cultured human microglia. Treatment of microglia with CCh (100 microM) produced a transient increase in [Ca2+]i, which was atropine-sensitive and was associated with release from intracellular Ca2+ stores. Successive applications of CCh showed a change in the amplitude of the [Ca2+]i signal consistent with desensitization. These results show that human microglia express functional muscarinic receptors and respond to cholinergic agonists. The rapid change of [Ca2+]i in microglia may serve as a second messenger to trigger downstream cascades which contribute to signalling pathways in CNS pathology.