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Neuroprotective role of phosphodiesterase inhibitor ibudilast on neuronal cell death induced by activated microglia
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Neuropharmacology 46 (2004) 404–411 www.elsevier.com/locate/neuropharm
Neuroprotective role of phosphodiesterase inhibitor ibudilast on neuronal cell death induced by activated microglia Tetsuya Mizuno a,∗, Tohru Kurotani b, Yukio Komatsu b, Jun Kawanokuchi a, Hideki Kato a, Norimasa Mitsuma a, Akio Suzumura a a b
Department of Neuroimmunology, Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan Department of Visual Neuroscience, Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8601, Japan Received 29 July 2003; received in revised form 8 September 2003; accepted 11 September 2003
Abstract The phosphodiesterase inhibitor, ibudilast, has many effects on lymphocytes, endothelial cells, and glial cells. We examined the neuroprotective role of ibudilast in neuron and microglia co-cultures. Ibudilast significantly suppressed neuronal cell death induced by the activation of microglia with lipopolysaccharide (LPS) and interferon (IFN)-γ. To examine the mechanisms by which ibudilast exerts a neuroprotective role against the activation of microglia, we examined the production of inflammatory and anti-inflammatory mediators and trophic factors following ibudilast treatment. In a dose-dependent manner, ibudilast suppressed the production of nitric oxide (NO), reactive oxygen species, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α and enhanced the production of the inhibitory cytokine, IL-10, and additional neurotrophic factors, including nerve growth factor (NGF), glia-derived neurotrophic factor (GDNF), and neurotrophin (NT)-4 in activated microglia. Thus, ibudilast-mediated neuroprotection was primarily due to the inhibition of inflammatory mediators and the upregulation of neurotrophic factor. In the CA1 region of hippocampal slices, longterm potentiation (LTP) induced by high frequency stimulation (HFS) could be inhibited with LPS and interferon-γ stimulation. Ibudilast returned this LTP inhibition to the levels observed in controls. These results suggest that ibudilast may be a useful neuroprotective and anti-dementia agent counteracting neurotoxicity in activated microglia. 2003 Elsevier Ltd. All rights reserved. Keywords: Ibudilast; Microglia; Nitric oxide; Tumor necrosis factor-α; Neuronal cell death; Long-term potentiation
1. Introduction Activated microglia function to exacerbate demyelinating diseases, such as multiple sclerosis and the animal model experimental allergic encephalomyelitis (EAE). These cells release proinflammatory mediators, including interleukin (IL)-1 (Giulian et al., 1986), IL-6 (Frei et al., 1989), tumor necrosis factor-α (TNF-α; Sawada et al., 1989; Wood, 1995), nitric oxide (NO; Meda et al., 1995), and reactive oxygen species (Tanaka et al., 1994). These molecular effectors act to destroy myelin or oligodendrocytes, producing demyelinating lesions (Selmaj and Raine, 1988; Selmaj et al., 1991). Cytotox-
Corresponding author. Tel.: +81-52-789-3881; fax: +81-52-7893885. E-mail address: [email protected] (T. Mizuno). ∗
0028-3908/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2003.09.009
icity of oligodendrocytes induced by activated microglia or macrophages is mediated by TNF-α (Zajicek et al., 1992). TNF-α can also cause neuronal cell death both directly and indirectly via the induction of NO and free radicals in glial cells (Hu et al., 1997). Activated microglia are found within the lesions of neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (ALS). Fibrillar forms of amyloid β peptide (Aβ) serve as an inflammatory stimulus of microglial lineage cells (Del Bo et al., 1995; Giulian et al., 1995). Exposure to a common herbicide, rotenone, induces features of parkinsonism. Rotenone stimulates the release of superoxide from microglia, resulting in selective destruction of the nigrostriatal dopaminergic system (Gao et al., 2002). In animal models, 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) damages the nigrostriatal dopaminergic pathway to induce Parkinson’s dis-
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ease. Neuronal damage induced by MPTP is mediated by activated microglia (Wu et al., 2002). An immunological mechanism of ALS pathogenesis has been proposed, following the identification of microglial activation within spinal cord gray matter and the motor cortex (Appel et al., 1994). Activated microglia facilitate TNF-α mediated motor neuron death in vitro (He et al., 2002). These observations suggested the active involvement of microglia in neurodegenerative disorders; agents that suppress microglial activation may be useful for future treatment of such pathological conditions. A cyclic AMP phosphodiesterase (PDE) inhibitor, ibudilast, has previously been used as an anti-asthma drug for its inhibitory effects on tracheal smooth muscle contractility (Kawasaki et al., 1992). Ibudilast has also been used for the treatment of stroke patients due to its platelet anti-aggregatory effects (Ohasi et al., 1986). PDE inhibitors suppress the production of TNF-α by various cells, also inhibiting EAE (Suzumura et al., 1999; Yoshikawa et al., 1999). The combination of a number of PDE inhibitors reduces the relapse rate of MS at well-tolerated therapeutic doses (Suzumura et al., 2000). In the present study, we investigated the suppression of activated microglia and the protection against microglia-induced neuronal cell death by ibudilast. We also investigated the effect of ibudilast on the long-term potentiation (LTP) of hippocampal CA1 neurons to evaluate the potential of ibudilast as an anti-dementia drug.
2. Materials and methods 2.1. Reagent Ibudilast was provided by Kyorin Pharmaceutical (Tokyo, Japan). Lipopolysaccharide (LPS) and NGmethyl-l-arginine (l-NMMA) were purchased from Sigma (St. Louis, MO, USA). Interferon (IFN)-γ and an anti-mouse TNF-α antibody were obtained from Genzyme/Techne (Minneapolis, MN, USA). 2.2. Cell cultures Microglia were isolated from primary mixed glial cell cultures from newborn STD-DDY mice on the 14th day by the “shaking off” method described previously (Suzumura et al., 1987). Cultures were 97–100% pure as determined by Fc receptor-specific immunostaining. Cultures were maintained in Eagle’s minimum essential medium supplemented with 10% fetal calf serum, 5 µg/ml bovine insulin, and 0.2% glucose. Neuron cultures were prepared from STD-DDY mice at embryonic day 17. Briefly, cortices were dissected, then freed of meninges. Cortical fragments were incu-
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bated with 0.25% trypsin and 20 µg/ml DNase I in phosphate-buffered saline at 37 °C for 15 min. Cortical fragments were then dissociated into single cells by pipetting, then resuspended in serum-free N2 medium. 2.3. Quantification of neuron survival Primary neuronal cells were plated in poly-ethyleneimine (PEI)-coated 24-well plates at a density of 2 × 105 cells per well. Microglia were plated in 2 cm2 cell culture inserts (membrane pore size 0.4 µm; Becton Dickinson) at a density of 2 × 105 cells per insert, then placed in the wells of neuronal cultures. Microglial insert cultures were stimulated with or without 1 µg/ml LPS and 100 ng/ml IFN-γ for 24 and 48 h, respectively, with a varied concentration of ibudilast. Viable neurons in the lower wells were then enumerated under a fluorescence microscope after staining with a viable cell marker, calcein AM (Molecular Probes). Similar cultures were also fixed with 4% paraformaldehyde and stained with Hoechst 33342 to visualize apoptotic cells. The cells containing condensed and fragmented nuclei were enumerated as above. 2.4. Measurement of cytokines, nitric oxide, superoxide anion and neurotrophic factors Supernatants from both the microglia upper culture inserts and the lower neuronal cultures were assessed for the levels of cytokines (IL-1β, IL-6, IL-10, and TNFα) and neurotrophic factors (nerve growth factor (NGF), BDNF, glia-derived neurotrophic factor (GDNF), neurotrophin (NT)-3, and NT-4) present using ELISA kits (Pharmingen). Measurement of NO was determined using a Griess reaction. Briefly, a 50 µl aliquot of the supernatant was incubated with an equal volume of Griess reagent (0.1% N-ethylenediamine dihydrochloride, 1% sulfanilamide, and 2.5% phosphoric acid) for 15 min at room temperature. The absorbance was then read at 540 nm in a microtiter plate reader. Nitrite concentrations were calculated from a standard curve of NaNO2 (Pollock et al., 1991). Superoxide anion formation was measured by NBT reduction assay as described (Suzumura et al., 1993). RNA was extracted from the remaining cells in the upper culture inserts to examine mRNA expression of cytokines, iNOS, and neurotrophic factors. 2.5. RNA extraction and reverse transcription-PCR Total RNA was extracted from microglia according to the guanidinium thiocyanate method (RNeasy Mini Kit; QIAGEN). cDNAs encoding mouse IL-1β, IL-6, NF-α, IL-10, NGF, BDNF, GDNF, NT-3, and NT-4 were generated by reverse transcription-PCR (RT-PCR) using Super Script II (Invitrogen) and Ampli Taq DNA poly-
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merase (Applied Biosystems) with the following primers: IL-1β sense, 5⬘-ATGGCAACTGTTCCTGAACTCAACT. IL-1β antisense, 5⬘-CAGGACAGGTATAGATTCTTT CCTTT. IL-6 sense, 5⬘-ATGAAGTTCCTCTCTGCAAGAGACT. IL-6 antisense, 5⬘-CACTAGGTTTGCCGAGTAGAT CTC. IL-10 sense, 5⬘-ATGCAGGACTTTAAGGGTTACTTG GGTT. IL-10 antisense, 5⬘-ATTTCGGAGAGAGGTACAAAC GAGGTTT. TNF-α sense, 5⬘-TTCTGTCTACTGAACTTCGGGGT GATCGGTCC. TNF-α antisense, 5⬘-GTATGAGATAGCAAATCG GCTGACGTGTGGG. NGF sense, 5⬘-CATGGGGGAGTTCTCAGTGT. NGF antisense, 5⬘-GCACCCACTCTCTCAACAGGAT. BDNF sense, 5⬘-GCGGCAGATAAAAAGACTGC. BDNF antisense, 5⬘-CTTATGAATCGCCAGCCAAT. GDNF sense, 5⬘-TATCCTGACCAGTTTGATGA. GDNF antisense, 5⬘-TCTAAAAACGACAGGTCGTC. NT-3 sense, 5⬘-TTTCTCGCTTATCTCCGTGGC. NT-3 antisense, 5⬘-AGGGTGCTCTGGTAATTTTCC. NT-4 sense, 5⬘-CCCTGCGTCAGTACTTCTTCGAGAC. NT-4 antisense, 5⬘-CTGGACGTCAGGCACGGCCTG TTC. 2.6. Induction of long-term potentiation Sprague Dawley rats (20–30 days postnatal) were first deeply anesthetized with isoflurane. The whole brain was then removed from the skull and immersed in an icecold oxygenated (95% O2 and 5% CO2) artificial cerebrospinal fluid (ACSF) containing 126 mM NaCl, 3 mM KCl, 1.3 mM MgSO4, 2.4 mM CaCl2, 1.2 mM NaH2PO4, 26 mM NaHCO3, and 10 mM glucose. Hippocampal slices (400 µm-thick) were prepared using a Microslicer (ZERO-1; Dosaka, Kyoto, Japan) and stored in an interface-type chamber perfused with ACSF at 33 °C. Two pairs of bipolar stimulating electrodes, made of tungsten wires (diameter, 100 µm; interpolar distance, 200 µm), were placed in the stratum radiatum of the CA1 region to stimulate Schaffer collateral/commissural pathways. Test stimulation (intensity 400–600 µA, duration 100 µ S at 0.1 Hz) was applied to s1 and s2 alternately at intervals of 5 s. An extracellular glass microelectrode was placed between the two stimulating electrodes to record the population excitatory postsynaptic potentials (EPSPs). To induce LTP, high frequency stimulation (HFS; 100 Hz, 1 s, two times separated by 10 s intervals) was applied five times to one of the electrodes at intervals of 5 min. The intensity of the test stimulation and HFS was adjusted to 25–30% of that eliciting the maximal responses. In the presence or
absence of ibudilast, 10 µg/ml LPS and 100 ng/ml IFNγ were added 60 min before HFS. LTP was then evaluated by measuring the changes in the initial slope of the population EPSPs. 2.7. Statistical analysis All experiments were performed in triplicate. For the statistical analyses, data were examined using the Student’s t-test. Statistical significance was indicated by a P value less than 0.05. 3. Results 3.1. Ibudilast attenuates activated microglia-induced neuronal cell death Unstimulated microglia did not exert any toxic effects on neuronal cells present at the bottom in neuron– microglial co-cultures (Fig. 1A). Stimulation of the upper microglial culture inserts with LPS (1 µg/ml) and IFN-γ (100 ng/ml), however, resulted in neuronal cell death exhibiting fragmented nuclei and shrunken cell bodies (Fig. 1B). Viable neurons in the lower wells decreased to 40% in 24 h and 20% in 48 h. The addition of 1, 10, or 100 µM ibudilast increased the rate of neuronal survival to 44%, 56%, and 74% in 24 h and 22%, 45%, and 68% in 48 h, respectively. The addition of 10 or 100 µM ibudilast significantly increased the neuronal survival rate (Figs. 1C and 2). By Hoechst 33342 staining (Fig. 1D–F), the number of apoptotic cells with condensed and fragmented nuclei increased in the neuronal cell population cultured with LPS and IFN-γ-activated microglia (Fig. 1E). The addition of ibudilast decreased the apoptotic changes observed in the neuronal cells in a dose-dependent manner (Fig. 1F). Ibudilast had no effect on the microglial population, and the addition of ibudilast to unstimulated microglia did not affect neuronal survival. To investigate the involvement of TNFα and NO in neuronal damage induced by activated microglia, we added either an anti-TNF-α antibody or the NO synthase inhibitor l-NMMA to activated microglial cultures. The neuronal survival rate at 48 h recovered to 42% of control levels following the addition of anti-TNF-α antibody, 45% after l-NMMA treatment, and 55% with concurrent administration of both antiTNF-α antibody and l-NMMA (Fig. 2B). The survival rates were lower, however, than those following ibudilast treatment. 3.2. Ibudilast inhibits proinflammatory cytokines, superoxide anion formation, and NO production by activated microglia To investigate the effect of ibudilast on radical formation by microglia, we examined the production of super-
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Fig. 1. Neuronal cell death by activated microglia and neuroprotection by ibudilast. In neuronal and microglial co-cultures, microglia in the upper cultures were stimulated by LPS (1 µg/ml) and IFN-γ (100 ng/ml) for 48 h in the presence or absence of ibudilast. (A–C) show phase contrast images, while (D–F) exhibit Hoechst staining. Cultures either (A, D) unstimulated or (B, E) stimulated with LPS (1 µg/ml) and IFN-γ (100 ng/ml) or following the (C, F) addition of ibudilast (100 µM) to activated microglia with LPS and IFN-γ. Data represent the mean ± SD from five independent experiments.
Fig. 2. Neuronal survival rates following ibudilast treatment, and the comparison of ibudilast, anti-TNF-α antibody and NO inhibitor, l-NMMA. Viability of untreated cells was set to 100%. (A) Neuronal survival rate for 24 (A) or 48 h (B). aT and LM designate anti-TNF-α antibody and l-NMMA treatment, respectively. Data represent the mean ± SD from five independent experiments.
oxides and NO by microglia in upper inserts stimulated with LPS (1 µg/ml) and IFN-γ (100 ng/ml). Ibudilast inhibited the production of these molecules, with significant suppression at 10 and 100 µM (Fig. 3A,B). To investigate the effect of ibudilast on proinflammatory cytokine production by activated microglia, we examined expression of IL-1β, IL-6, and TNF-α at the mRNA and protein levels. Ibudilast suppressed both IL-1β and IL-6 production at 100 µM, and significantly suppressed TNF-α production at 10 and 100 µM. The mRNA expression levels of these cytokines also decreased in a dose-dependent manner (Fig. 4). Ibudilast thus has a strong anti-inflammatory effect.
3.3. Ibudilast induces production of an inhibitory cytokine, IL-10, and the neurotrophic factors, GDNF and NT-4, by activated microglia We next investigated the effect of ibudilast on antiinflammatory cytokine production. The anti-inflammatory cytokine IL-10 is induced by activated microglia following LPS treatment (Mizuno et al., 1994). Ibudilast upregulated IL-10 production in a dose-dependent manner (Fig. 5A). We then tested the production of the neurotrophic factors, NGF, BDNF, GDNF, NT-3, and NT-4. Ibudilast increased NGF mRNA and protein levels (Fig. 5B) and GDNF and NT-4 mRNA expression. GDNF and
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considered a model of learning and memory. In the control LTP (without LPS/IFN-γ stimulation), the level of EPSPs is maintained at 100% at 180 min. Ibudilast itself did not affect the control LTP. The addition of 10 µg/ml LPS and 100 ng/ml IFN-γ 60 min before HFS disturbed the maintenance of late phase LTP. The magnitude of LTP decreased to 80% of the induction level, 180 min after HFS (Fig. 6A). The addition of 100 µM ibudilast at LPS and IFN-γ stimulation returned LTP to normal levels (Fig. 6B).
Fig. 3. The effect of ibudilast on superoxide and NO production by activated microglia. Microglia in upper wells were treated with LPS (1 µg/ml) and IFN-γ (100 ng/ml) for 48h. Data indicating (A) superoxide formation and (B) NO production represent the mean ± SD from five independent experiments.
NT-4 protein levels were below those detectable by our assay (data not shown). BDNF and NT-3 were not upregulated by ibudilast treatment (Fig. 5C). 3.4. Ibudilast recovers the disturbance of hippocampal LTP induced with LPS and IFN-g To investigate the function of ibudilast within the central nervous system, especially in learning and memory, we examined the effect of ibudilast on hippocampal LTP. LTP in the hippocampal CA1 region is an activitydependent enhancement of synaptic efficacy, which is
4. Discussion Ibudilast is a type IV, X specific PDE inhibitor (MacKenzie et al., pers. comm.). In this study, we demonstrated that ibudilast suppressed the neuronal cell death induced by activated microglia. Activated microglia release a variety of proinflammatory cytokines, NO, peroxynitrite, and reactive oxygen species, all of which are toxic to neuronal cells. While TNF-α is the major neurotoxic agent secreted by β-amyloidstimulated microglia (Combs et al., 2001), peroxynitrite, which is formed by NO and superoxides, may also play a significant role in neurotoxicity (Xie et al., 2002). Using an anti-TNF-α antibody and the NO synthase inhibitor l-NMMA, we investigated the role of TNFα and NO on neuronal cell death induced by activated microglia. The neuronal survival rate of cultures treated with an anti-TNF-α antibody and l-NMMA were lower
Fig. 4. The effect of ibudilast on proinflammatory cytokine production by activated microglia. Microglia in upper well cultures were treated with LPS (1 µg/ml) and IFN-γ (100 ng/ml) for 24 h. The proinflammatory cytokines (A) TNF-α, (B) IL-1β, and (C) IL-6 were evaluated by EIA (top panel) and RT-PCR (bottom panel). Data represent the mean ± SD from five independent experiments.
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Fig. 5. Upregulation of the anti-inflammatory cytokine, IL-10, and neurotrophic factors by ibudilast. Microglia in upper well cultures were treated with LPS (1 µg/ml) and IFN-γ (100 ng/ml) for 24 h. IL-10 and NGF were evaluated by EIA (top panel) and RT-PCR (bottom panel). (A) IL-10, (B) NGF, (C) BDNF, GDNF, NT-3, and NT-4 mRNA expression. Data represent the mean ± SD from five independent experiments.
Fig. 6. Effect of ibudilast on LTP suppression with LPS and IFN-γ. (A) The mean population EPSP slope after tetanic stimulation was gradually attenuated with LPS (1 µg/ml) and IFN-γ (100 ng/ml). (B) The addition of 100 µM ibudilast with LPS and IFN-γ stimulation recovered LTP to normal levels. Data represent the mean ± SE from four independent experiments. The horizontal line indicates control level at unstimulated sites.
than those treated with ibudilast, suggesting that ibudilast may suppress additional neurotoxic pathways. Ibudilast suppressed TNF-α and NO as well as the additional IL-1β and IL-6, and upregulated the anti-inflammatory cytokine, IL-10. In addition, ibudilast treatment also enhanced the production of neurotrophic factors, such as NGF, GDNF, and NT-4. IL-1β is also neurotoxic, mediating ischemic injury in rat retinal ganglion cells (Yoneda et al., 2001). Although IL-6 promotes the survival, differentiation, and growth of neurons, IL-6 has recently been shown to reduce hippocampal neurogen-
esis (Vallieres et al., 2002). Thus, suppression of IL-1 and IL-6 may also contribute to the favorable effects of ibudilast observed on neuronal cell survival. The induction of IL-10 may also contribute to the neuronal survival by suppressing TNF-α, IL-1, and IL-6 (Mizuno et al., 1994; Kambayashi et al., 1995). Apoptotic PC 12 cells promote the production of NGF and TGF-β by microglia (De Simone et al., 2003). GDNF inhibits the expression of NO synthetase during ischemia (Wang et al., 1997). Thus, the upregulation of neurotrophic factors by ibudilast may also support neuronal cell survival. There are various subtype-specific PDE inhibitors, including pentoxifylline, a nonselective PDE inhibitor that blocks the release of TNF-α from microglia (Chao et al., 1992; Suzumura et al., 1998). Pentoxifylline does not, however, affect nitrite accumulation, providing only minor protection against activated microglia (Xie et al., 2002). Vesnarinone, a type III PDE inhibitor, suppresses TNF-α, IL-1β and IL-6, but does not induce IL-10 (Kambayashi et al., 1995). Thus, ibudilast suppresses microglial activation more effectively than other PDE inhibitors. PDE inhibitors have been reported to increase cytoplasmic cAMP, resulting in the downregulation of nuclear factor NF-B and the upregulation of cAMP responsive element-binding protein (CREB) (Parry and Mackman, 1997) via phosphorylation by protein kinase A (PKA). The suppression of either the activation and/or translocation of nuclear factor NF-B results in the depression of proinflammatory cytokines production and iNOS expression. The upregulation of CREB is thus crucial for neuronal survival.
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PKA/CREB pathway is also important in LTP, widely recognized to be essential for memory acquisition. Late stage LTP in hippocampal CA1 neurons depends heavily on this pathway (Frey et al., 1993). Amyloid β peptide 1–42 (Aβ1–42) treatment of cultured hippocampal neurons reportedly leads to the inactivation of PKA/CREB pathway and inhibition of LTP. The PDE inhibitor, rolipram, reverses this inhibition (Vitolo et al., 2002). In our experiments, the addition of LPS and IFN-γ before LTP induction can activate microglia on hippocampal slices, allowing us to investigate the effect of activated microglia on LTP. Following stimulation with LPS and IFN-γ, the magnitude of LTP decreased gradually, indicating that LTP is disturbed by the activation of microglia. Ibudilast reverses this inhibition, suggesting the therapeutic potential of ibudilast as an antidementia drug. In the AD brain, increased levels of TNF-α inducible nitric oxide synthase, and the peroxynitrite marker, nitrotyrosine, have been reported (Hensley et al., 1998; Tarkowski et al., 1999). This elevation may be due to the activation of microglia. Although we used LPS and IFNγ to activate microglia in this study, ibudilast also suppressed the neurotoxic effectors of microglia stimulated with Aβ1–42 (unpublished observation). As ibudilast is now widely and safely used to treat patients with stroke or asthma in Japan, this drug may be useful in the treatment of both neurodegenerative disorders, such as AD, and ischemic disorders in which microglial activation is a critical step in pathogenesis.
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Neuropharmacology 46 (2004) 404–411 www.elsevier.com/locate/neuropharm
Neuroprotective role of phosphodiesterase inhibitor ibudilast on neuronal cell death induced by activated microglia Tetsuya Mizuno a,∗, Tohru Kurotani b, Yukio Komatsu b, Jun Kawanokuchi a, Hideki Kato a, Norimasa Mitsuma a, Akio Suzumura a a b
Department of Neuroimmunology, Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan Department of Visual Neuroscience, Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8601, Japan Received 29 July 2003; received in revised form 8 September 2003; accepted 11 September 2003
Abstract The phosphodiesterase inhibitor, ibudilast, has many effects on lymphocytes, endothelial cells, and glial cells. We examined the neuroprotective role of ibudilast in neuron and microglia co-cultures. Ibudilast significantly suppressed neuronal cell death induced by the activation of microglia with lipopolysaccharide (LPS) and interferon (IFN)-γ. To examine the mechanisms by which ibudilast exerts a neuroprotective role against the activation of microglia, we examined the production of inflammatory and anti-inflammatory mediators and trophic factors following ibudilast treatment. In a dose-dependent manner, ibudilast suppressed the production of nitric oxide (NO), reactive oxygen species, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α and enhanced the production of the inhibitory cytokine, IL-10, and additional neurotrophic factors, including nerve growth factor (NGF), glia-derived neurotrophic factor (GDNF), and neurotrophin (NT)-4 in activated microglia. Thus, ibudilast-mediated neuroprotection was primarily due to the inhibition of inflammatory mediators and the upregulation of neurotrophic factor. In the CA1 region of hippocampal slices, longterm potentiation (LTP) induced by high frequency stimulation (HFS) could be inhibited with LPS and interferon-γ stimulation. Ibudilast returned this LTP inhibition to the levels observed in controls. These results suggest that ibudilast may be a useful neuroprotective and anti-dementia agent counteracting neurotoxicity in activated microglia. 2003 Elsevier Ltd. All rights reserved. Keywords: Ibudilast; Microglia; Nitric oxide; Tumor necrosis factor-α; Neuronal cell death; Long-term potentiation
1. Introduction Activated microglia function to exacerbate demyelinating diseases, such as multiple sclerosis and the animal model experimental allergic encephalomyelitis (EAE). These cells release proinflammatory mediators, including interleukin (IL)-1 (Giulian et al., 1986), IL-6 (Frei et al., 1989), tumor necrosis factor-α (TNF-α; Sawada et al., 1989; Wood, 1995), nitric oxide (NO; Meda et al., 1995), and reactive oxygen species (Tanaka et al., 1994). These molecular effectors act to destroy myelin or oligodendrocytes, producing demyelinating lesions (Selmaj and Raine, 1988; Selmaj et al., 1991). Cytotox-
Corresponding author. Tel.: +81-52-789-3881; fax: +81-52-7893885. E-mail address: [email protected] (T. Mizuno). ∗
0028-3908/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2003.09.009
icity of oligodendrocytes induced by activated microglia or macrophages is mediated by TNF-α (Zajicek et al., 1992). TNF-α can also cause neuronal cell death both directly and indirectly via the induction of NO and free radicals in glial cells (Hu et al., 1997). Activated microglia are found within the lesions of neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (ALS). Fibrillar forms of amyloid β peptide (Aβ) serve as an inflammatory stimulus of microglial lineage cells (Del Bo et al., 1995; Giulian et al., 1995). Exposure to a common herbicide, rotenone, induces features of parkinsonism. Rotenone stimulates the release of superoxide from microglia, resulting in selective destruction of the nigrostriatal dopaminergic system (Gao et al., 2002). In animal models, 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) damages the nigrostriatal dopaminergic pathway to induce Parkinson’s dis-
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ease. Neuronal damage induced by MPTP is mediated by activated microglia (Wu et al., 2002). An immunological mechanism of ALS pathogenesis has been proposed, following the identification of microglial activation within spinal cord gray matter and the motor cortex (Appel et al., 1994). Activated microglia facilitate TNF-α mediated motor neuron death in vitro (He et al., 2002). These observations suggested the active involvement of microglia in neurodegenerative disorders; agents that suppress microglial activation may be useful for future treatment of such pathological conditions. A cyclic AMP phosphodiesterase (PDE) inhibitor, ibudilast, has previously been used as an anti-asthma drug for its inhibitory effects on tracheal smooth muscle contractility (Kawasaki et al., 1992). Ibudilast has also been used for the treatment of stroke patients due to its platelet anti-aggregatory effects (Ohasi et al., 1986). PDE inhibitors suppress the production of TNF-α by various cells, also inhibiting EAE (Suzumura et al., 1999; Yoshikawa et al., 1999). The combination of a number of PDE inhibitors reduces the relapse rate of MS at well-tolerated therapeutic doses (Suzumura et al., 2000). In the present study, we investigated the suppression of activated microglia and the protection against microglia-induced neuronal cell death by ibudilast. We also investigated the effect of ibudilast on the long-term potentiation (LTP) of hippocampal CA1 neurons to evaluate the potential of ibudilast as an anti-dementia drug.
2. Materials and methods 2.1. Reagent Ibudilast was provided by Kyorin Pharmaceutical (Tokyo, Japan). Lipopolysaccharide (LPS) and NGmethyl-l-arginine (l-NMMA) were purchased from Sigma (St. Louis, MO, USA). Interferon (IFN)-γ and an anti-mouse TNF-α antibody were obtained from Genzyme/Techne (Minneapolis, MN, USA). 2.2. Cell cultures Microglia were isolated from primary mixed glial cell cultures from newborn STD-DDY mice on the 14th day by the “shaking off” method described previously (Suzumura et al., 1987). Cultures were 97–100% pure as determined by Fc receptor-specific immunostaining. Cultures were maintained in Eagle’s minimum essential medium supplemented with 10% fetal calf serum, 5 µg/ml bovine insulin, and 0.2% glucose. Neuron cultures were prepared from STD-DDY mice at embryonic day 17. Briefly, cortices were dissected, then freed of meninges. Cortical fragments were incu-
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bated with 0.25% trypsin and 20 µg/ml DNase I in phosphate-buffered saline at 37 °C for 15 min. Cortical fragments were then dissociated into single cells by pipetting, then resuspended in serum-free N2 medium. 2.3. Quantification of neuron survival Primary neuronal cells were plated in poly-ethyleneimine (PEI)-coated 24-well plates at a density of 2 × 105 cells per well. Microglia were plated in 2 cm2 cell culture inserts (membrane pore size 0.4 µm; Becton Dickinson) at a density of 2 × 105 cells per insert, then placed in the wells of neuronal cultures. Microglial insert cultures were stimulated with or without 1 µg/ml LPS and 100 ng/ml IFN-γ for 24 and 48 h, respectively, with a varied concentration of ibudilast. Viable neurons in the lower wells were then enumerated under a fluorescence microscope after staining with a viable cell marker, calcein AM (Molecular Probes). Similar cultures were also fixed with 4% paraformaldehyde and stained with Hoechst 33342 to visualize apoptotic cells. The cells containing condensed and fragmented nuclei were enumerated as above. 2.4. Measurement of cytokines, nitric oxide, superoxide anion and neurotrophic factors Supernatants from both the microglia upper culture inserts and the lower neuronal cultures were assessed for the levels of cytokines (IL-1β, IL-6, IL-10, and TNFα) and neurotrophic factors (nerve growth factor (NGF), BDNF, glia-derived neurotrophic factor (GDNF), neurotrophin (NT)-3, and NT-4) present using ELISA kits (Pharmingen). Measurement of NO was determined using a Griess reaction. Briefly, a 50 µl aliquot of the supernatant was incubated with an equal volume of Griess reagent (0.1% N-ethylenediamine dihydrochloride, 1% sulfanilamide, and 2.5% phosphoric acid) for 15 min at room temperature. The absorbance was then read at 540 nm in a microtiter plate reader. Nitrite concentrations were calculated from a standard curve of NaNO2 (Pollock et al., 1991). Superoxide anion formation was measured by NBT reduction assay as described (Suzumura et al., 1993). RNA was extracted from the remaining cells in the upper culture inserts to examine mRNA expression of cytokines, iNOS, and neurotrophic factors. 2.5. RNA extraction and reverse transcription-PCR Total RNA was extracted from microglia according to the guanidinium thiocyanate method (RNeasy Mini Kit; QIAGEN). cDNAs encoding mouse IL-1β, IL-6, NF-α, IL-10, NGF, BDNF, GDNF, NT-3, and NT-4 were generated by reverse transcription-PCR (RT-PCR) using Super Script II (Invitrogen) and Ampli Taq DNA poly-
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merase (Applied Biosystems) with the following primers: IL-1β sense, 5⬘-ATGGCAACTGTTCCTGAACTCAACT. IL-1β antisense, 5⬘-CAGGACAGGTATAGATTCTTT CCTTT. IL-6 sense, 5⬘-ATGAAGTTCCTCTCTGCAAGAGACT. IL-6 antisense, 5⬘-CACTAGGTTTGCCGAGTAGAT CTC. IL-10 sense, 5⬘-ATGCAGGACTTTAAGGGTTACTTG GGTT. IL-10 antisense, 5⬘-ATTTCGGAGAGAGGTACAAAC GAGGTTT. TNF-α sense, 5⬘-TTCTGTCTACTGAACTTCGGGGT GATCGGTCC. TNF-α antisense, 5⬘-GTATGAGATAGCAAATCG GCTGACGTGTGGG. NGF sense, 5⬘-CATGGGGGAGTTCTCAGTGT. NGF antisense, 5⬘-GCACCCACTCTCTCAACAGGAT. BDNF sense, 5⬘-GCGGCAGATAAAAAGACTGC. BDNF antisense, 5⬘-CTTATGAATCGCCAGCCAAT. GDNF sense, 5⬘-TATCCTGACCAGTTTGATGA. GDNF antisense, 5⬘-TCTAAAAACGACAGGTCGTC. NT-3 sense, 5⬘-TTTCTCGCTTATCTCCGTGGC. NT-3 antisense, 5⬘-AGGGTGCTCTGGTAATTTTCC. NT-4 sense, 5⬘-CCCTGCGTCAGTACTTCTTCGAGAC. NT-4 antisense, 5⬘-CTGGACGTCAGGCACGGCCTG TTC. 2.6. Induction of long-term potentiation Sprague Dawley rats (20–30 days postnatal) were first deeply anesthetized with isoflurane. The whole brain was then removed from the skull and immersed in an icecold oxygenated (95% O2 and 5% CO2) artificial cerebrospinal fluid (ACSF) containing 126 mM NaCl, 3 mM KCl, 1.3 mM MgSO4, 2.4 mM CaCl2, 1.2 mM NaH2PO4, 26 mM NaHCO3, and 10 mM glucose. Hippocampal slices (400 µm-thick) were prepared using a Microslicer (ZERO-1; Dosaka, Kyoto, Japan) and stored in an interface-type chamber perfused with ACSF at 33 °C. Two pairs of bipolar stimulating electrodes, made of tungsten wires (diameter, 100 µm; interpolar distance, 200 µm), were placed in the stratum radiatum of the CA1 region to stimulate Schaffer collateral/commissural pathways. Test stimulation (intensity 400–600 µA, duration 100 µ S at 0.1 Hz) was applied to s1 and s2 alternately at intervals of 5 s. An extracellular glass microelectrode was placed between the two stimulating electrodes to record the population excitatory postsynaptic potentials (EPSPs). To induce LTP, high frequency stimulation (HFS; 100 Hz, 1 s, two times separated by 10 s intervals) was applied five times to one of the electrodes at intervals of 5 min. The intensity of the test stimulation and HFS was adjusted to 25–30% of that eliciting the maximal responses. In the presence or
absence of ibudilast, 10 µg/ml LPS and 100 ng/ml IFNγ were added 60 min before HFS. LTP was then evaluated by measuring the changes in the initial slope of the population EPSPs. 2.7. Statistical analysis All experiments were performed in triplicate. For the statistical analyses, data were examined using the Student’s t-test. Statistical significance was indicated by a P value less than 0.05. 3. Results 3.1. Ibudilast attenuates activated microglia-induced neuronal cell death Unstimulated microglia did not exert any toxic effects on neuronal cells present at the bottom in neuron– microglial co-cultures (Fig. 1A). Stimulation of the upper microglial culture inserts with LPS (1 µg/ml) and IFN-γ (100 ng/ml), however, resulted in neuronal cell death exhibiting fragmented nuclei and shrunken cell bodies (Fig. 1B). Viable neurons in the lower wells decreased to 40% in 24 h and 20% in 48 h. The addition of 1, 10, or 100 µM ibudilast increased the rate of neuronal survival to 44%, 56%, and 74% in 24 h and 22%, 45%, and 68% in 48 h, respectively. The addition of 10 or 100 µM ibudilast significantly increased the neuronal survival rate (Figs. 1C and 2). By Hoechst 33342 staining (Fig. 1D–F), the number of apoptotic cells with condensed and fragmented nuclei increased in the neuronal cell population cultured with LPS and IFN-γ-activated microglia (Fig. 1E). The addition of ibudilast decreased the apoptotic changes observed in the neuronal cells in a dose-dependent manner (Fig. 1F). Ibudilast had no effect on the microglial population, and the addition of ibudilast to unstimulated microglia did not affect neuronal survival. To investigate the involvement of TNFα and NO in neuronal damage induced by activated microglia, we added either an anti-TNF-α antibody or the NO synthase inhibitor l-NMMA to activated microglial cultures. The neuronal survival rate at 48 h recovered to 42% of control levels following the addition of anti-TNF-α antibody, 45% after l-NMMA treatment, and 55% with concurrent administration of both antiTNF-α antibody and l-NMMA (Fig. 2B). The survival rates were lower, however, than those following ibudilast treatment. 3.2. Ibudilast inhibits proinflammatory cytokines, superoxide anion formation, and NO production by activated microglia To investigate the effect of ibudilast on radical formation by microglia, we examined the production of super-
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Fig. 1. Neuronal cell death by activated microglia and neuroprotection by ibudilast. In neuronal and microglial co-cultures, microglia in the upper cultures were stimulated by LPS (1 µg/ml) and IFN-γ (100 ng/ml) for 48 h in the presence or absence of ibudilast. (A–C) show phase contrast images, while (D–F) exhibit Hoechst staining. Cultures either (A, D) unstimulated or (B, E) stimulated with LPS (1 µg/ml) and IFN-γ (100 ng/ml) or following the (C, F) addition of ibudilast (100 µM) to activated microglia with LPS and IFN-γ. Data represent the mean ± SD from five independent experiments.
Fig. 2. Neuronal survival rates following ibudilast treatment, and the comparison of ibudilast, anti-TNF-α antibody and NO inhibitor, l-NMMA. Viability of untreated cells was set to 100%. (A) Neuronal survival rate for 24 (A) or 48 h (B). aT and LM designate anti-TNF-α antibody and l-NMMA treatment, respectively. Data represent the mean ± SD from five independent experiments.
oxides and NO by microglia in upper inserts stimulated with LPS (1 µg/ml) and IFN-γ (100 ng/ml). Ibudilast inhibited the production of these molecules, with significant suppression at 10 and 100 µM (Fig. 3A,B). To investigate the effect of ibudilast on proinflammatory cytokine production by activated microglia, we examined expression of IL-1β, IL-6, and TNF-α at the mRNA and protein levels. Ibudilast suppressed both IL-1β and IL-6 production at 100 µM, and significantly suppressed TNF-α production at 10 and 100 µM. The mRNA expression levels of these cytokines also decreased in a dose-dependent manner (Fig. 4). Ibudilast thus has a strong anti-inflammatory effect.
3.3. Ibudilast induces production of an inhibitory cytokine, IL-10, and the neurotrophic factors, GDNF and NT-4, by activated microglia We next investigated the effect of ibudilast on antiinflammatory cytokine production. The anti-inflammatory cytokine IL-10 is induced by activated microglia following LPS treatment (Mizuno et al., 1994). Ibudilast upregulated IL-10 production in a dose-dependent manner (Fig. 5A). We then tested the production of the neurotrophic factors, NGF, BDNF, GDNF, NT-3, and NT-4. Ibudilast increased NGF mRNA and protein levels (Fig. 5B) and GDNF and NT-4 mRNA expression. GDNF and
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considered a model of learning and memory. In the control LTP (without LPS/IFN-γ stimulation), the level of EPSPs is maintained at 100% at 180 min. Ibudilast itself did not affect the control LTP. The addition of 10 µg/ml LPS and 100 ng/ml IFN-γ 60 min before HFS disturbed the maintenance of late phase LTP. The magnitude of LTP decreased to 80% of the induction level, 180 min after HFS (Fig. 6A). The addition of 100 µM ibudilast at LPS and IFN-γ stimulation returned LTP to normal levels (Fig. 6B).
Fig. 3. The effect of ibudilast on superoxide and NO production by activated microglia. Microglia in upper wells were treated with LPS (1 µg/ml) and IFN-γ (100 ng/ml) for 48h. Data indicating (A) superoxide formation and (B) NO production represent the mean ± SD from five independent experiments.
NT-4 protein levels were below those detectable by our assay (data not shown). BDNF and NT-3 were not upregulated by ibudilast treatment (Fig. 5C). 3.4. Ibudilast recovers the disturbance of hippocampal LTP induced with LPS and IFN-g To investigate the function of ibudilast within the central nervous system, especially in learning and memory, we examined the effect of ibudilast on hippocampal LTP. LTP in the hippocampal CA1 region is an activitydependent enhancement of synaptic efficacy, which is
4. Discussion Ibudilast is a type IV, X specific PDE inhibitor (MacKenzie et al., pers. comm.). In this study, we demonstrated that ibudilast suppressed the neuronal cell death induced by activated microglia. Activated microglia release a variety of proinflammatory cytokines, NO, peroxynitrite, and reactive oxygen species, all of which are toxic to neuronal cells. While TNF-α is the major neurotoxic agent secreted by β-amyloidstimulated microglia (Combs et al., 2001), peroxynitrite, which is formed by NO and superoxides, may also play a significant role in neurotoxicity (Xie et al., 2002). Using an anti-TNF-α antibody and the NO synthase inhibitor l-NMMA, we investigated the role of TNFα and NO on neuronal cell death induced by activated microglia. The neuronal survival rate of cultures treated with an anti-TNF-α antibody and l-NMMA were lower
Fig. 4. The effect of ibudilast on proinflammatory cytokine production by activated microglia. Microglia in upper well cultures were treated with LPS (1 µg/ml) and IFN-γ (100 ng/ml) for 24 h. The proinflammatory cytokines (A) TNF-α, (B) IL-1β, and (C) IL-6 were evaluated by EIA (top panel) and RT-PCR (bottom panel). Data represent the mean ± SD from five independent experiments.
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Fig. 5. Upregulation of the anti-inflammatory cytokine, IL-10, and neurotrophic factors by ibudilast. Microglia in upper well cultures were treated with LPS (1 µg/ml) and IFN-γ (100 ng/ml) for 24 h. IL-10 and NGF were evaluated by EIA (top panel) and RT-PCR (bottom panel). (A) IL-10, (B) NGF, (C) BDNF, GDNF, NT-3, and NT-4 mRNA expression. Data represent the mean ± SD from five independent experiments.
Fig. 6. Effect of ibudilast on LTP suppression with LPS and IFN-γ. (A) The mean population EPSP slope after tetanic stimulation was gradually attenuated with LPS (1 µg/ml) and IFN-γ (100 ng/ml). (B) The addition of 100 µM ibudilast with LPS and IFN-γ stimulation recovered LTP to normal levels. Data represent the mean ± SE from four independent experiments. The horizontal line indicates control level at unstimulated sites.
than those treated with ibudilast, suggesting that ibudilast may suppress additional neurotoxic pathways. Ibudilast suppressed TNF-α and NO as well as the additional IL-1β and IL-6, and upregulated the anti-inflammatory cytokine, IL-10. In addition, ibudilast treatment also enhanced the production of neurotrophic factors, such as NGF, GDNF, and NT-4. IL-1β is also neurotoxic, mediating ischemic injury in rat retinal ganglion cells (Yoneda et al., 2001). Although IL-6 promotes the survival, differentiation, and growth of neurons, IL-6 has recently been shown to reduce hippocampal neurogen-
esis (Vallieres et al., 2002). Thus, suppression of IL-1 and IL-6 may also contribute to the favorable effects of ibudilast observed on neuronal cell survival. The induction of IL-10 may also contribute to the neuronal survival by suppressing TNF-α, IL-1, and IL-6 (Mizuno et al., 1994; Kambayashi et al., 1995). Apoptotic PC 12 cells promote the production of NGF and TGF-β by microglia (De Simone et al., 2003). GDNF inhibits the expression of NO synthetase during ischemia (Wang et al., 1997). Thus, the upregulation of neurotrophic factors by ibudilast may also support neuronal cell survival. There are various subtype-specific PDE inhibitors, including pentoxifylline, a nonselective PDE inhibitor that blocks the release of TNF-α from microglia (Chao et al., 1992; Suzumura et al., 1998). Pentoxifylline does not, however, affect nitrite accumulation, providing only minor protection against activated microglia (Xie et al., 2002). Vesnarinone, a type III PDE inhibitor, suppresses TNF-α, IL-1β and IL-6, but does not induce IL-10 (Kambayashi et al., 1995). Thus, ibudilast suppresses microglial activation more effectively than other PDE inhibitors. PDE inhibitors have been reported to increase cytoplasmic cAMP, resulting in the downregulation of nuclear factor NF-B and the upregulation of cAMP responsive element-binding protein (CREB) (Parry and Mackman, 1997) via phosphorylation by protein kinase A (PKA). The suppression of either the activation and/or translocation of nuclear factor NF-B results in the depression of proinflammatory cytokines production and iNOS expression. The upregulation of CREB is thus crucial for neuronal survival.
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PKA/CREB pathway is also important in LTP, widely recognized to be essential for memory acquisition. Late stage LTP in hippocampal CA1 neurons depends heavily on this pathway (Frey et al., 1993). Amyloid β peptide 1–42 (Aβ1–42) treatment of cultured hippocampal neurons reportedly leads to the inactivation of PKA/CREB pathway and inhibition of LTP. The PDE inhibitor, rolipram, reverses this inhibition (Vitolo et al., 2002). In our experiments, the addition of LPS and IFN-γ before LTP induction can activate microglia on hippocampal slices, allowing us to investigate the effect of activated microglia on LTP. Following stimulation with LPS and IFN-γ, the magnitude of LTP decreased gradually, indicating that LTP is disturbed by the activation of microglia. Ibudilast reverses this inhibition, suggesting the therapeutic potential of ibudilast as an antidementia drug. In the AD brain, increased levels of TNF-α inducible nitric oxide synthase, and the peroxynitrite marker, nitrotyrosine, have been reported (Hensley et al., 1998; Tarkowski et al., 1999). This elevation may be due to the activation of microglia. Although we used LPS and IFNγ to activate microglia in this study, ibudilast also suppressed the neurotoxic effectors of microglia stimulated with Aβ1–42 (unpublished observation). As ibudilast is now widely and safely used to treat patients with stroke or asthma in Japan, this drug may be useful in the treatment of both neurodegenerative disorders, such as AD, and ischemic disorders in which microglial activation is a critical step in pathogenesis.
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