Akio Suzumura

Tumor Necrosis Factor-α Induces Neurotoxicity via Glutamate Release from Hemichannels of Activated Microglia in an Autocrine Manner

Glutamate released by activated microglia induces excitoneurotoxicity and may contribute to neuronal damage in neurodegenerative diseases, including Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, and multiple sclerosis. In addition, tumor necrosis factor-α (TNF-α) secreted from activated microglia may elicit neurodegeneration through caspase-dependent cascades and silencing cell survival signals. However, direct neurotoxicity of TNF-α is relatively weak, because TNF-α also increases production of neuroprotective factors. Accordingly, it is still controversial how TNF-α exerts neurotoxicity in neurodegenerative diseases. Here we have shown that TNF-α is the key cytokine that stimulates extensive microglial glutamate release in an autocrine manner by up-regulating glutaminase to cause excitoneurotoxicity. Further, we have demonstrated that the connexin 32 hemichannel of the gap junction is another main source of glutamate release from microglia besides glutamate transporters. Although pharmacological blockade of glutamate receptors is a promising therapeutic candidate for neurodegenerative diseases, the associated perturbation of physiological glutamate signals has severe adverse side effects. The unique mechanism of microglial glutamate release that we describe here is another potential therapeutic target. We rescued neuronal cell death in vitro by using a glutaminase inhibitor or hemichannel blockers to diminish microglial glutamate release without perturbing the physiological glutamate level. These drugs may give us a new therapeutic strategy against neurodegenerative diseases with minimum adverse side effects.

Expression of cytokine receptors in cultured neuronal and glial cells

We investigated mRNA expression of cytokine receptors in three different types of glial cells and two neuronal line cells by the RT-PCR method. Microglia expressed mRNA for receptors of IL-3, -4, -6, -7, GM-CSF, and M-CSF. Astrocytes were positive for receptors of IL-6, -7, GM-CSF, and M-CSF. Oligodendrocytes were positive for receptors of IL-3, -4, -7, GM-CSF, and M-CSF. Neuronal cells expressed receptors of IL-6 and GM-CSF with very low levels. This is the first demonstration of cytokine receptor mRNA expression in isolated glial and neuronal cells.

Expression of glutamate transporters in cultured glial cells

Expression of mRNAs for glutamate transporter (GLT-1) and glutamate aspartate transporter (GLAST) was investigated in three different types of purified glial cells by the reverse transcriptase-polymerase chain reaction (RT-PCR). Cultured astrocytes, oligodendrocytes, and microglia expressed mRNAs for GLAST and GLT-1; mRNA for GLAST was expressed more prominently than that for GLT-1 in astrocytes. Oligodendrocytes and microglia expressed mRNAs for both GLT-1 and GLAST equally, but the expression in microglia was not prominent, suggesting glutamate uptake is not essential in microglia. In astrocytes cultured from different brain regions, GLAST mRNA was equally expressed. GLT-1 mRNA was also detected in these astrocytes, but the expression level was lower than that of GLAST.

Cytokine network in the central nervous system and its roles in growth and differentiation of glial and neuronal cells

Cells resident within the central nervous system (CNS) can synthesize, secrete and respond to inflammatory cytokines not only contributing to the responses to injury or immunological challenge within the CNS, but also regulating their own growth and differentiation potential. The actions and cell communication via cytokines in the CNS are designated as the CNS cytokine network, in which microglia and astrocytes play the central roles. To further characterize the CNS cytokine network we investigated the differences in roles of these cells, and found that microglia might contribute to the early phase of cytokine production reaction and that astrocytes might contribute the late phase of the reaction. We also investigated roles of inhibitory cytokines such as TGF β, IL‐4, and IL‐10, and showed that each might play a distinct role in the inhibitory regulation in the CNS. We summarized our previous report about cellular distribution of cytokine receptors in the CNS cells and discussed their roles in the CNS cytokine network. Finally, we investigated that expression of IL‐6 and IL‐2 receptors in neuronal and oligodendrocytic differentiation, respectively. From these results, we discussed the features of the CNS cytokine network.

Interferon-γ directly induces neurotoxicity through a neuron specific, calcium-permeable complex of IFN-γ receptor and AMPA GluR1 receptor

Interferon‐γ (IFN‐γ) is a proinflamma tory cytokine that plays a pivotal role in pathology of diseases in the central nervous system (CNS), such as multiple sclerosis. However, the direct effect of IFN‐γ on neuronal cells has yet to be elucidated. We show here that IFN‐γ directly induces neuronal dysfunction, which appears as dendritic bead formation in mouse cortical neurons and enhances glutamate neurotoxicity mediated via alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isox‐ azolepropionic (AMPA) receptors but not N‐methyl‐D‐ aspartate receptors. In the CNS, IFN‐γ receptor forms a unique, neuron‐specific, calcium‐permeable receptor complex with AMPA receptor subunit GluRl. Through this receptor complex, IFN‐γ phosphorylates GluRl at serine 845 position by JAKT2/STAT1 pathway, in creases Ca2+ influx and following nitric oxide production, and subsequently decreases ATP production, leading to the dendritic bead formation. These findings provide novel mechanisms of neuronal excitotoxicity, which may occur in both inflammatory and neurodegen erative diseases in the CNS.— Mizuno, T., Zhang, G., Takeuchi, H., Kawanokuchi, J., Wang, J., Sonobe, Y., Jin, S., Takada, N., Komatsu, Y., Suzumura, A. Inter feron‐γ directly induces neurotoxicity through a neu ron specific, calcium‐ permeable complex of IFN‐γ receptor and AMPA GluRl receptor. FASEB J. 22, 1797–1806 (2008)

Production of interferon-γ by microglia

Neural cells do not usually interact with immune cells because of the lack of major histocompatibility complex (MHC) antigen expression. Interferon-γ (IFN-γ) enables this interaction via induction of MHC antigen expression in neural cells. Thus, IFN-γ is a critical cytokine for the development of central nervous system (CNS) pathologies. IFN-γ, however, is considered to be produced exclusively by lymphoid cells. Here, we show for the first time that murine microglia produce IFN-γ in response to IL-12 and/or IL-18, using RT-PCR detection of IFN-γ mRNA and Western blotting and immunohistochemical analysis for cytoplasmic expression of IFN-γ. Stimulation of microglia with IL-12 and IL-18 resulted in MHC class II mRNA expression in microglia. Since IL-12 and IL-18 are produced in the CNS by glial cells, these cytokines may play a critical role in the initiation of neural immune cell interaction and the induction of autoimmune processes in the CNS via induction of IFN-γ and MHC antigens.

Effects of phosphodiesterase inhibitors on cytokine production by microglia

Type III and IV phosphodiesterase inhibitors (PDEIs) have recently been shown to suppress the production of TNF-α in several types of cells. In the present study, we have shown that all the types of PDEIs, from type 1- to V-specific and non-specific, suppress the production of TNF-α by mouse microglia stimulated with lipopolysaccharide (LPS) in a dose-dependent manner. Certain combinations of three different types of PDEIs synergistically suppressed TNF-α production by microglia at a very low concentration (1 μM). Since some PDEIs reportedly pass through the blood-brain barrier (BBB), the combination of three PDEIs may be worth trying in neurological diseases, such as multiple sclerosis and HIV-related neurological diseases in which TNF-α may play a critical role. Some PDEIs also suppressed interleukin-1 (IL-1) and IL-6 production by mouse microglia stimulated with LPS. In contrast, the production of IL-10, which is known to be an inhibitory cytokine, was upregulated by certain PDEIs. The suppression of TNF-α and induction of IL-10 were confirmed at the mRNA level by RT - PCR. PDEIs may be useful anti-inflammatory agents by downregulating inflammatory cytokines and upregulating inhibitory cytokines in the central nervous system. (CNS).

Interleukin-4 induces proliferation and activation of microglia but suppresses their induction of class II major histocompatibility complex antigen expression

Abstract We recently found that microglia, brain macrophages, express interleukin-4 (IL-4) receptor mRNA in vitro. Since IL-4 exhibits a variety of functions on the cells of monocyte-macrophage lineage, we examined the effects of IL-4on the functions of microglia. Recombinant IL-4 induced the proliferation of microglia in a dose- and time-dependent manner as determined by MTT colorimetric assay, [3H]thymidine uptake and bromodeoxyuridine (BrdU) incorporation. IL-4 also synergistically enhanced the proliferation of microglia with such colony-stimulating factors as IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF). It also increased acid phosphatase activity and superoxide anion formation by these cells. Despite these positive effects on proliferation and activation, IL-4 suppressed the IFN γ-induced class II MHC antigen expression in these cells. Since these effects of recombinant IL-4 inhibited by the addition of monoclonal antibody against IL-4 receptors, the effects of IL-4 on microglia appear to be a specific function via IL-4 receptors. Although microglia and astrocytes produce a variety of immunoregulatory cytokines, neither cell produced IL-4 as determined by bioassay or detection of IL-4 mRNA by RT-PCR method. Thus, the exogenous IL-4 may contribute to the accumulation of microglia in or around inflammatory lesions in the central nervous system, and may be involved in the regulatory mechanisms of microglia.

Expression of cytokines during glial differentiation.

Astrocytes and microglia produce a variety of cytokines, some of which may have roles in the proliferation and differentiation of glial cells during development in the central nervous system. Cytokine mRNAs and activities were therefore assayed during glial development in mixed glial cell cultures from newborn mouse brain. Cytokine mRNAs were also measured in mouse brain during postnatal development in vivo. Macrophage colony-stimulating factor(M-CSF) mRNA, interleukin-1 beta (IL-1 beta) mRNA and tumor necrosis factor alpha (TNF alpha) mRNA were all detected on the in vitro cultures and each showed a distinct time course of expression. IL-6 and granulocyte-macrophage colony-stimulating factor(GM-CSF) mRNAs were not detected in the cultured cells. Measurements of cytokine activity in culture supernatants as well as cytokine mRNAs in vivo gave similar results. The data suggest that IL-1, TNF alpha and M-CSF are produced in the period of gliogenesis, and that M-CSF rather than GM-CSF may promote the generation and proliferation of microglia. Although IL-6 and GM-CSF exhibit neurotrophic effects, these cytokines may not function as neurotrophic factors during early postnatal development.

Interleukin-25 expressed by brain capillary endothelial cells maintains blood-brain barrier function in a protein kinase Cepsilon-dependent manner.

Interleukin (IL)-25, a member of the IL-17 family of cytokines, is expressed in the brains of normal mice. However, the cellular source of IL-25 and its function in the brain remain to be elucidated. Here, we show that IL-25 plays an important role in preventing infiltration of the inflammatory cells into the central nervous system. Brain capillary endothelial cells (BCECs) express IL-25. However, it is down-regulated by inflammatory cytokines, including tumor necrosis factor (TNF)-α, IL-17, interferon-γ, IL-1β, and IL-6 in vitro, and is also reduced in active multiple sclerosis (MS) lesions and in the inflamed spinal cord of experimental autoimmune encephalomyelitis, an animal model of MS. Furthermore, IL-25 restores the reduced expression of tight junction proteins, occludin, junction adhesion molecule, and claudin-5, induced by TNF-α in BCECs and consequently repairs TNF-α-induced blood-brain barrier (BBB) permeability. IL-25 induces protein kinase Cϵ (PKCϵ) phosphorylation, and up-regulation of claudin-5 is suppressed by PKCϵ inhibitor peptide in the IL-25-stimulated BCECs. These results suggest that IL-25 is produced by BCECs and protects against inflammatory cytokine-induced excessive BBB collapse through a PKCϵ-dependent pathway. These novel functions of IL-25 in maintaining BBB integrity may help us understand the pathophysiology of inflammatory brain diseases such as MS.