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Role of hippocampal TLR4 in neurotoxicity in mice following toluene exposure
Neurotoxicology and Teratology 33 (2011) 598–602
Contents lists available at ScienceDirect
Neurotoxicology and Teratology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / n e u t e r a
Brief communication
Role of hippocampal TLR4 in neurotoxicity in mice following toluene exposure Tin-Tin Win-Shwe a, Naoki Kunugita b, Yasuhiro Yoshida c, Hidekazu Fujimaki a,⁎ a b c
National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan National Institute of Public Health, 2-3-6 Minami, Wako-shi, Saitama, 351-0197, Japan University of Occupational and Environmental Health, Japan, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu, Fukuoka 807-8555, Japan
a r t i c l e
i n f o
Article history: Received 1 June 2011 Received in revised form 8 July 2011 Accepted 12 July 2011 Available online 23 July 2011 Keywords: Toll-like receptor Toluene Hippocampus Mice
a b s t r a c t The present study was designed to investigate the possible involvement of TLR4 pathway in the mouse hippocampus following toluene exposure. Male C3H/HeN and C3H/HeJ (TLR4 defective) mice were exposed to 0, 5, 50 or 500 ppm of toluene for 6 weeks. The expressions of TLR4-related signal transduction pathway mRNAs in the hippocampi were examined using real-time RT-PCR and an immunohistochemical analysis. In C3H/HeN mice, the relative mRNA expression levels of TLR4 and NF-κB activating protein were significantly up-regulated in the groups exposed to toluene, but not in the C3H/HeJ mice. Heat shock protein 70, a possible endogenous ligand for TLR4, mRNA was increased in the C3H/HeN mice exposed to toluene. This is the first report to show that TLR4 may have a role in the neurotoxic effects in mice exposed to toluene. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Individuals with different immunogenetic backgrounds have different sensitivities to toxic chemical exposure. Previously, we investigated the possible role of the major histocompatibility complex (MHC) locus at the neurotransmitter level in the hippocampi of congenic mice (C57BL/10 [H2b] and B10.BR/Sg [H-2k]) following toluene exposure. We found different patterns of glutamate, taurine and glycine levels in both the control and the toluene-treated mice (Win-Shwe et al., 2009). Recently, our laboratory reported that the role of strain differences in sensitivity to low-level toluene exposure on neurotrophins and their receptor levels in the mouse hippocampus in three mouse strains (C3H/HeN, BALB/c and C57BL/10) with or without allergic stimulation. We found that low-level toluene exposure may induce the up-regulation of neurotrophin-related gene expression in the mouse hippocampus, depending on the mouse strain, and that allergic stimulation in sensitive strains may decrease the threshold for sensitivity to a lower exposure level (Win-Shwe et al., 2010a). Toluene, a volatile organic solvent, is widely used in industry and a number of commercial products such as cosmetics, inks, adhesive, paints and glues. Toluene is a well-known neurotoxicant; however, the mechanism of action of toluene has not yet been clarified. Innate immunity is the primary response to protect the body from microorganisms that are identified through molecular patterns recognized by TLRs. Among the TLRs, TLR4 is expressed on a wide ⁎ Corresponding author at: Research Center for Environmental Risk National Institute for Environmental Studies 16-2, Onogawa, Tsukuba, Ibaraki 305-8506, Japan. Tel.: + 81 29 850 2518; fax: + 81 29 850 2518. E-mail address: [email protected] (H. Fujimaki). 0892-0362/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ntt.2011.07.005
range of nervous system cell types, such as microglia (Glezer et al., 2007; Olson and Miller, 2004), astrocytes (Bsibsi et al., 2006; Farina et al., 2005), oligodendrocytes (Bsibsi et al., 2002), and neurons (Lafon et al., 2006). TLR4 is crucial for the identification of lipopolysaccharides (LPS) present in the cell wall component of Gram-negative bacteria (Hoshino et al., 1999; Poltorak et al., 1998). The binding of LPS to its cognate TLR4 triggers the NF-κB transduction pathway, leading to the transcriptional activation of proinflammatory cytokines, chemokines and other genes essential for pathogen elimination (Akira et al., 2001; Beutler, 2002). TLRs might not only serve to sense microbial pathogen-associated molecular patterns (PAMPs), but also to sense certain endogenous factors that act as damage-associated molecular pattern (DAMP) molecules (Rubartelli and Lotze, 2007). Potential DAMP molecules include heat shock proteins, fibrinogen, fibronectin, heparin sulfate, soluble hyaluronan, oxidized LDL, gangliosides, fatty acids and other cues of dying cells, and these molecules can stimulate TLR4 and TLR2 signaling (Marshak-Rothstein, 2006; Beg, 2002; McMahon et al., 2005). In a brain cell injury model, the activation of TLR4 and NF-κB may be neuroprotective, either increasing cell resistance or removing toxic molecules via an increase in the phagocytic capacity of activated microglia (Glezer et al., 2006). However, little is known about the role of TLR4 in neurogenesis following environmental exposure to toxic chemicals. Previously, we reported that NMDA receptor subunit and related protein kinase and transcription factor may act as neurological biomarkers in toluene-induced developmental-phase specific neurotoxicity (Win-Shwe et al., 2010b). Moreover, we demonstrated that neurotrophins and proinflammatory cytokines are important
T.-T. Win-Shwe et al. / Neurotoxicology and Teratology 33 (2011) 598–602
neuroinflammatory mediators via TLR4-dependent NF-κB pathway in toluene-induced developmental-phase specific immunotoxicity (Win-Shwe et al., 2011). From these two studies, we suggest that a late postnatal period of mouse which corresponds with the third trimester in human may be the susceptible/critical window for toluene exposure in developmental toxicity. Our previous studies prompted us to investigate the sensitive biomarkers in toluene-induced neurotoxicity in adult mice. Thus, in the present study, we hypothesized that toluene exposure may induce neuronal damage and release DAMP molecules from damaged neurons in the mouse hippocampus, resulting the activation of TLR4 signaling pathways. These molecules may act as endogenous ligands that bind to TLR4, triggering the production of proinflammatory cytokines via the activation of NF-κB or other transcription factors and inducing neuroinflammation and neurodegeneration. In the presence of TLR4, neurons and glia cells are activated and take part in a compensatory brain repair mechanism. Therefore, in the present study, using wild type and TLR4 defective mice, we investigated the possible involvement of hippocampal TLR4 in neurotoxicity following toluene exposure. 2. Materials and methods 2.1. Animals Eight-week-old male C3H/HeN and C3H/HeJ mice were obtained from Charles River Japan Inc. (Yokohama, Japan) and were used at 10 weeks of age. Food and water were given ad libitum. This study was approved by the Ethics Committee for Experimental Animals of the University of Occupational and Environmental Health, Japan. 2.2. Exposure to toluene Toluene vapor was generated using an organic solvent gas generator (Shibata Scientific Technology Ltd.), diluted with clean, filtered air to achieve the desired gas concentrations, and then introduced into a stainless steel and glass chamber as described previously (Hori et al., 1999). The toluene concentration in the chamber was monitored using gas chromatography (Model 353B; GL Sciences, Tokyo, Japan). The average toluene levels (mean ± SD) for the 5, 50, or 500-ppm experiments were 4.9 ± 0.4, 50.5 ± 2.0, and 505 ± 11.8 ppm, respectively. Each group of mice (n= 9) was exposed to a filtered air control (0 ppm) or toluene for 6 h (from 10:00 to 16:00 h) per day, 5 days per week for 6 weeks. 2.3. Quantification of the hippocampal mRNA expression levels One day following the final toluene inhalation, the mice were sacrificed under deep pentobarbital anesthesia and the hippocampi were collected from each group of mice (n = 6) and frozen quickly in liquid nitrogen, then stored at − 80 °C until the total RNA was extracted. Briefly, total RNA extraction from the hippocampal samples was performed using the BioRobot EZ-1 and EZ-1 RNA tissue mini kits (Qiagen GmbH, Germany). Then, the purity of the total RNA was examined, and the quantity was estimated using the ND-1000 NanoDrop RNA Assay protocol (NanoDrop, USA), as described previously (Win-Shwe et al., 2007). Next, we performed first-strand cDNA synthesis from the total RNA using SuperScript RNase H − Reverse Transcriptase II (Invitrogen, USA), according to the manufacturer's protocol. Next we examined mRNA expression of 18S, TLR4, NF-κB activating protein (NF-κB), hyaluronan synthase 1(Has1), and heat shock protein 70 (Hspa-1 α) by real-time RT-PCR (Applied Biosystems Inc., USA). The tissue 18S rRNA level was used as an internal control. The primer sequences for 18S were forward 5′TACCACATCCAAGAAGGCAG-3′, reverse 5′-TGCCCTCCAATGGATCCTC3′ and TLR4 were forward 5′-AAGGCATGGCATGGCTTACAC-3′, reverse
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5′-GGCCAATTTTGTCTCCACAGC-3′. Some primers (NF-κB, ID_67050; Has1, ID_15116; and Hspa-1 α, ID_193740) were purchased from Qiagen, Sample & Assay Technologies (Qiagen GmbH, Germany). Then, the relative expression levels of the TLR4-related transduction pathway mRNAs were individually normalized to the 18S rRNA content in the respective samples and expressed as mRNA signals per unit of 18S rRNA expression. 2.4. Immunohistochemistry On the day after the final toluene exposure, the brains were removed from three C3H/HeN and C3H/HeJ mice from each of the control and toluene-exposed groups after the animals had been deeply anesthetized with sodium pentobarbital; the brains were then fixed with 10% formalin. The fixed brains were dehydrated using a graded series of ethanol, cleared with xylene, and embedded in paraffin. Coronal paraffin sections were cut at a thickness of 10 μm using a cryostat and were mounted on 3-aminopropyltriethoxysilanecoated glass slides. Iba1 (microglia marker) was detected immunohistochemically in the hippocampus. Briefly, deparaffinized sections were immersed in absolute ethanol followed by 10% H2O2 for 10 min each at room temperature. After rinsing in 0.01-M phosphate buffer saline, the sections were blocked with 2% normal swine serum in PBS for 30 min at room temperature and then reacted with rabbit anti-Iba1 (diluted 1:250; Wako Pure Chemical Industries, Ltd; Osaka, Japan) in PBS for 1 h at 37 °C. The sections were reacted with biotinylated donkey anti-rabbit IgG (1:300 Histofine; Nichirei Bioscience, Tokyo, Japan) in PBS for 1 h at 37 °C before and after rinsing in PBS. The sections were then incubated with peroxidase streptavidin (1:300, ABC KIT) containing PBS for 1 h at room temperature. After rinsing in PBS, Iba1 immunoreactivity was detected using a Dako DAB Plus Liquid System (DAKO). To determine the immunoreactivity of Iba1 in the hippocampus, photomicrographic digital images (150 dpi, 256 scales) of the hippocampal regions were taken using a CCD camera connected to a light microscope. 2.5. Statistical analysis All the data were expressed as the mean ± standard error (S.E.). The statistical analysis was performed using the StatMate II statistical analysis system for Microsoft Excel, Version 5.0 (Nankodo Inc., Tokyo, Japan). The dose–response data were analyzed using a one-way analysis of variance with a post-hoc analysis using the Bonferroni/ Dunn method. Then, data within each group was further analyzed using the Student's t-test. Differences were considered to be significant at P b 0.05. 3. Results We investigated the effect of various concentrations of toluene on the relative mRNA expression of TLR4 and compared the effects in wild-type TLR4-possessing C3H/HeN mice with those in TLR4-mutant C3H/HeJ mice to evaluate the involvement of TLR signaling. As shown in Fig. 1 (A), exposure to 50 ppm of toluene significantly increased the relative mRNA expression of TLR4 in C3H/HeN mice. There is no significant change observed between the control and the 500 ppm toluene-exposed groups. On the other hand, no increase in the relative mRNA expression of TLR4 was observed in C3H/HeJ mice (Fig. 1 (B)). TLR4 signaling ultimately leads to the induction of NF-κB, followed by the production of proinflammatory cytokines and the upregulation of cell surface molecules involved in the initiation of adaptive immune responses to pathogens (Olson and Miller, 2004). In C3H/HeN mice, the relative mRNA expression level of NF-κB activating protein was increased in the 50 ppm toluene-exposed group (P b 0.05, Fig. 1 (C)). However, exposure to 500 ppm toluene significantly suppressed the relative mRNA expression of NF-κB
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C Relative mRNA expression NF-κB/18S
Relative mRNA expression TLR4/18S
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Fig. 1. Relative mRNA expression of TLR4 and NF-κB mRNAs in the hippocampi of C3H/HeN and C3H/HeJ mice. Relative mRNA expression of TLR4 in (A) C3H/HeN, (B) C3H/HeJ mice and NF-κB mRNA in (C) C3H/HeN, (D) C3H/HeJ mice following exposure to 0, 5, 50 or 500 ppm of toluene for 6 weeks. Each bar represents the mean ± SE (n = 6) (*P b 0.05).
Relative mRNA expression Hspa1-α α/18S
activating protein. In the C3H/HeJ mice, however, no significant changes were observed between the control and toluene-exposed groups (Fig. 1 (D)). Our results suggested an absence of NF-κB activation in TLR4-defective mice. Hyaluronan and heat shock protein are potential DAMP molecules or endogenous ligands for TLR4. To detect the role of TLR4 in tolueneinduced neuroinflammation, we examined the relative mRNA expression level of hyaluronan (Has1) and heat shock protein 70 (Hspa1-α) in the hippocampi of C3H/HeN and C3H/HeJ mice. The expression of Has1 mRNA was not detected in either the C3H/HeN or the C3H/HeJ mice (data not shown). In the C3H/HeN mice, the relative mRNA expression level of Hspa1-α was increased in the tolueneexposed group (P b 0.05, Fig. 2). However, in the C3H/HeJ mice, no significant difference was observed between 0 and 50 ppm toluene exposed mice. We also detected the major immune cell microglia in the hippocampi of C3H/HeN and C3H/HeJ mice after toluene exposure. We found that microglia activation was prominent in the hippocam-
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Fig. 2. Relative mRNA expression of Hspa1-α mRNA in the hippocampi of C3H/HeN and C3H/HeJ mice.Relative mRNA expression of Hapa1 in C3H/HeN and C3H/HeJ mice following exposure to 0 or 50 ppm of toluene for 6 weeks. Each bar represents the mean ± SE (n = 6) (*P b 0.05).
pal area of toluene-exposed C3H/HeN (Fig. 3 (A)) and C3H/HeJ (Fig. 3 (B)) mice, compared with corresponding control mice. Moreover, toluene-exposed C3H/HeN mice showed more prominent microglia than C3H/HeJ mice. 4. Discussion Rapidly accumulating evidence suggests that environmental toxic chemicals may cause adverse health effects by disrupting the homeostatic regulatory mechanisms of the nervous and immune systems. Toluene is a well-known neurotoxicant, and previous data from both in vivo and in vitro animal studies support the hippocampus as a target for toluene (Korbo et al., 1996; Terashi et al., 1997; Gelazonia et al., 2006). In Japan, the occupational exposure limit for toluene is 50 ppm (Japan Society for Occupational Health, 1994), while the recently updated threshold limit in the United States is 20 ppm (ACGIH, 2006). TLR4 is primarily regarded as an innate immune receptor (Takeda et al., 2003); thus, its function in the brain has been commonly attributed to neuroimmune responses. TLRs recognize patterns, rather than specific molecules, in addition to their ability to recognize physiological compounds (Ohashi et al., 2000; Okamura et al., 2001; Johnson et al., 2003). TLRs also have an innate ability to mediate a rapid response to a wide range of signals in the microenvironment, and not merely to pathogens. Both in vitro and in vivo studies have indicated that TLR4 signaling may contribute to pathogenetic changes arising from environmental exposure to toxic chemicals. A previous in vitro study stated that alveolar macrophages produce proinflammatory cytokines, such as TNF-α and IL-6, in response to airborne particulates through TLR2 and 4 (Becker et al., 2002). Moreover, the induction of airway hyperresponsiveness after ozone inhalation can reportedly occur through TLR4 (Hollingsworth et al., 2004; Garantziotis et al., 2010). In vivo studies have also shown that TLR4 is necessary for airway inflammation induced by diesel exhaust particles (Takano et al., 2002; Inoue et al., 2006). Although the role of TLR4
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Fig. 3. Microglial morphology in the hippocampus of C3H/HeN and C3H/HeJ mice.Representative digital photomicrographs of Iba1-immunostained sections taken from the hippocampal region in (A) C3H/HeN and (B) C3H/HeJ mice following exposure to toluene at 0 ppm or 50 ppm for 6 weeks (scale bar = 50 μm).
signaling in airway pathogenesis has been extensively demonstrated, the role of TLR4 in brain function after environmental toxic chemical exposure remains unclear. The present study showed that exposure to toluene may induce neuroinflammation via the TLR4–NF-κB signaling pathway, in which heat shock protein may be a potential endogenous ligand for TLR4. Instead of a dose-dependent pattern, we observed a reverse U-shaped curve for the TLR4–NF-κB induced inflammatory response in C3H/ HeN mice. No mechanism has been proposed to explain the above stimulatory effect of low levels of toluene exposure, but one possibility is that toluene inhalation contributes to neural dysfunction related to sensory stimulation, and that neurotoxic reaction and homeostatic mechanisms compensate for toxicant-induced changes (Win-Shwe and Fujimaki, 2010c). We also found that microglia activation were more prominent in toluene-exposed C3H/HeN mice than in C3H/HeJ mice. We suggest that toluene may induce DAMP molecules, such as heat shock protein, in the hippocampi of C3H/HeN mice. The induction of such molecules would, in turn, stimulate TLR4 signaling and lead to the production of proinflammatory cytokines via NF-κB-dependent or -independent pathways, resulting in neuroinflammation and leading to neuron and glia activation. Neuroprotective TLR signaling might involve both immune cells and nonimmune cells, such as glia-producing neurotrophic factors (Hanisch et al., 2008). In addition, recent evidence has indicated that TLR4 plays a role in the behavioral consequences of alcohol-induced inflammatory damage via activation of microglia and astrocyte, and induction of cytokines and inflammatory mediators in mice (Pascual et al., 2011). In conclusion, we observed different regulatory mechanisms in TLR4 signaling pathway in C3H/HeN and C3H/HeJ mice exposed to low-level toluene. Finally, our previous and present studies and other studies suggest that TLR4 and related signaling pathway may act as
potential sensitive biomarkers in neurotoxicity induced by environmental toxic chemical exposure. Conflict of interest statement All authors declare that there are no conflicts of interest. Acknowledgments This research was partly supported by a grant from the Ministry of Education, Culture, Sports, Sciences and Technology, Japan (#21390198) to H. F. We are grateful to Dr. K. Arashidani for supporting toluene exposure and Ms. K. Ohnishi, K. Taki and S. Hirayama for their technical assistance. References Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol 2001;2:675–80. American Conference of Governmental Industrial Hygienists (ACGIH). Threshold limit values and biological exposure indices guide. www.acgih.org/TLV/Studies.htm 2006. Becker S, Fenton MJ, Soukup JM. Involvement of microbial components and toll-like receptors 2 and 4 in cytokine responses to air pollution particles. Am J Respir Cell Mol Biol 2002;27:611–8. Beg A. Endogenous ligands of Toll-like receptors: implications for regulating inflammatory and immune responses. Trends Immunol 2002;23:509–12. Beutler B. Toll-like receptors: how they work and what they do. Curr Opin Hematol 2002;9:2–10. Bsibsi M, Ravid R, Gveric D, van Noort JM. Broad expression of Toll-like receptors in the human central nervous system. J Neuropathol Exp Neurol 2002;61:1013–21. Bsibsi M, Persoon-Deen C, Verwer RW, Meeuwsen S, Ravid R, Van Noort JM. Toll-like receptor 3 on adult human astrocytes triggers production of neuroprotective mediators. Glia 2006;53:688–95. Farina C, Krumbholz M, Giese T, Hartmann G, Aloisi F, Meinl E. Preferential expression and function of Toll-like receptor 3 in human astrocytes. J Neuroimmunol 2005;159:12–9.
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Garantziotis S, Li Z, Potts EN, Lindsey JY, Stober VP, Polosukhin VV, et al. TLR4 is necessary for hyaluronan-mediated airway hyperresponsiveness after ozone inhalation. Am J Respir Crit Care Med 2010;181:666–75. Gelazonia L, Japaridze N, Svanidze I. Pyramidal cell loss in hippocampus of young rats exposed to toluene. Georgian Med News 2006;135:126–8. Glezer I, Zekki H, Scavone C, Rivest S. Modulation of the innate immune response by NMDA receptors has neuropathological consequences. J Neurosci 2006;23: 11094–103. Glezer I, Simard AR, Rivest S. Neuroprotective role of the innate immune system by microglia. Neuroscience 2007;147:867–83. Hanisch UK, Johnson TV, Kipnis J. Toll-like receptors: roles in neuroprotection? Trends Neurosci 2008;31:176–82. Hollingsworth 2nd JW, Cook DN, Brass DM, Walker JK, Morgan DL, Foster WM, et al. The role of Toll-like receptor 4 in environmental airway injury in mice. Am J Respir Crit Care Med 2004;170:126–32. Hori H, Ishidao T, Oyabu T, Yamato H, Morimoto Y, Tanaka I. Effect of simultaneous exposure to methanol and toluene vapor on their metabolites in rats. J Occup Health 1999;41:149–53. Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, Takeda Y, et al. Cutting edge: Tolllike receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol 1999;162:3749–52. Inoue K, Takano H, Yanagisawa R, Hirano S, Ichinose T, Shimada A, et al. The role of tolllike receptor 4 in airway inflammation induced by diesel exhaust particles. Arch Toxicol 2006;80:275–9. Japan Society for Occupational Health. Toluene. Ind Med 1994;36:103–10. Johnson GB, Brunn GJ, Platt JL. Activation of mammalian Toll-like receptors by endogenous agonists. Crit Rev Immunol 2003;23:15–44. Korbo L, Ladefoged O, Lam HR, Ostergaard G, West MJ, Arlien-Søborg P. Neuronal loss in hippocampus in rats exposed to toluene. Neurotoxicology 1996;17:359–66. Lafon M, Megret F, Lafage M, Prehaud C. The innate immune facet of brain: human neurons express TLR-3 and sense viral dsRNA. J Mol Neurosci 2006;29:185–94. Marshak-Rothstein A. Toll-like receptors in systemic autoimmune disease. Nat Rev Immunol 2006;6:823–35. McMahon EJ, Bailey SL, Castenada CV, Waldner H, Miller SD. Epitope spreading initiates in the CNS in two mouse models of multiple sclerosis. Nat Med 2005;11:335–9. Ohashi K, Burkart V, Flohé S, Kolb H. Cutting edge: heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex. J Immunol 2000;164:558–61.
Okamura Y, Watari M, Jerud ES, Young DW, Ishizaka ST, Rose J, et al. The extra domain A of fibronectin activates Toll-like receptor 4. J Biol Chem 2001;276:10229–33. Olson JK, Miller SD. Microglia initiate central nervous system innate and adaptive immune responses through multiple TLRs. J Immunol 2004;173:3916–24. Pascual M, Baliño P, Alfonso-Loeches S, Aragón CM, Guerri C. Impact of TLR4 on behavioral and cognitive dysfunctions associated with alcohol-induced neuroinflammatory damage. Brain Behav Immun 2011;25(Suppl 1):S80–91. Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 1998;282: 2085–8. Rubartelli A, Lotze MT. Inside, outside, upside down: damage-associated molecularpattern molecules (DAMPs) and redox. Trends Immunol 2007;28:429–36. Takano H, Yanagisawa R, Ichinose T, Sadakane K, Yoshino S, Yoshikawa T, et al. Diesel exhaust particles enhance lung injury related to bacterial endotoxin through expression of proinflammatory cytokines, chemokines, and intercellular adhesion molecule-1. Am J Respir Crit Care Med 2002;165:1329–35. Takeda K, Kaisho T, Akira S. Toll-like receptors. Annu Rev Immunol 2003;21:335–76. Terashi H, Nagata K, Satoh Y, Hirata Y, Hatazawa J. Hippocampal hypoperfusion underlying dementia due to chronic toluene intoxication. Rinsho Shinkeigaku 1997;37:1010–3. Win-Shwe TT, Yamamoto S, Nakajima D, Furuyama A, Fukushima A, Ahmed S, et al. Modulation of neurological related allergic reaction in mice exposed to low-level toluene. Toxicol Appl Pharmacol 2007;222:17–24. Win-Shwe TT, Mitsushima D, Yamamoto S, Funabashi T, Fujimaki H. Strain differences in extracellular amino acid neurotransmitter levels in the hippocampi of major histocompatibility complex congenic mice in response to toluene exposure. Neuroimmunomodulation 2009;16:185–90. Win-Shwe TT, Tsukahara S, Yamamoto S, Fukushima A, Kunugita N, Arashidani K, et al. Up-regulation of neurotrophin-related gene expression in mouse hippocampus following low-level toluene exposure. Neurotoxicology 2010a;31:85–93. Win-Shwe TT, Yoshida Y, Kunugita N, Tsukahara S, Fujimaki H. Does early life toluene exposure alter the expression of NMDA receptor subunits and signal transduction pathway in infant mouse hippocampus? Neurotoxicology 2010b;31:647–53. Win-Shwe TT, Fujimaki H. Neurotoxicity of toluene. Toxicol Lett 2010c;198:93–9. Win-Shwe TT, Kunugita N, Yoshida Y, Nakajima D, Tsukahara S, Fujimaki H. Differential mRNA expression of neuroimmune markers in the hippocampus of infant mice following toluene exposure during brain developmental period. J Appl Toxicol 2011. doi:10.1002/jat.1643. Mar 5.
Contents lists available at ScienceDirect
Neurotoxicology and Teratology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / n e u t e r a
Brief communication
Role of hippocampal TLR4 in neurotoxicity in mice following toluene exposure Tin-Tin Win-Shwe a, Naoki Kunugita b, Yasuhiro Yoshida c, Hidekazu Fujimaki a,⁎ a b c
National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan National Institute of Public Health, 2-3-6 Minami, Wako-shi, Saitama, 351-0197, Japan University of Occupational and Environmental Health, Japan, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu, Fukuoka 807-8555, Japan
a r t i c l e
i n f o
Article history: Received 1 June 2011 Received in revised form 8 July 2011 Accepted 12 July 2011 Available online 23 July 2011 Keywords: Toll-like receptor Toluene Hippocampus Mice
a b s t r a c t The present study was designed to investigate the possible involvement of TLR4 pathway in the mouse hippocampus following toluene exposure. Male C3H/HeN and C3H/HeJ (TLR4 defective) mice were exposed to 0, 5, 50 or 500 ppm of toluene for 6 weeks. The expressions of TLR4-related signal transduction pathway mRNAs in the hippocampi were examined using real-time RT-PCR and an immunohistochemical analysis. In C3H/HeN mice, the relative mRNA expression levels of TLR4 and NF-κB activating protein were significantly up-regulated in the groups exposed to toluene, but not in the C3H/HeJ mice. Heat shock protein 70, a possible endogenous ligand for TLR4, mRNA was increased in the C3H/HeN mice exposed to toluene. This is the first report to show that TLR4 may have a role in the neurotoxic effects in mice exposed to toluene. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Individuals with different immunogenetic backgrounds have different sensitivities to toxic chemical exposure. Previously, we investigated the possible role of the major histocompatibility complex (MHC) locus at the neurotransmitter level in the hippocampi of congenic mice (C57BL/10 [H2b] and B10.BR/Sg [H-2k]) following toluene exposure. We found different patterns of glutamate, taurine and glycine levels in both the control and the toluene-treated mice (Win-Shwe et al., 2009). Recently, our laboratory reported that the role of strain differences in sensitivity to low-level toluene exposure on neurotrophins and their receptor levels in the mouse hippocampus in three mouse strains (C3H/HeN, BALB/c and C57BL/10) with or without allergic stimulation. We found that low-level toluene exposure may induce the up-regulation of neurotrophin-related gene expression in the mouse hippocampus, depending on the mouse strain, and that allergic stimulation in sensitive strains may decrease the threshold for sensitivity to a lower exposure level (Win-Shwe et al., 2010a). Toluene, a volatile organic solvent, is widely used in industry and a number of commercial products such as cosmetics, inks, adhesive, paints and glues. Toluene is a well-known neurotoxicant; however, the mechanism of action of toluene has not yet been clarified. Innate immunity is the primary response to protect the body from microorganisms that are identified through molecular patterns recognized by TLRs. Among the TLRs, TLR4 is expressed on a wide ⁎ Corresponding author at: Research Center for Environmental Risk National Institute for Environmental Studies 16-2, Onogawa, Tsukuba, Ibaraki 305-8506, Japan. Tel.: + 81 29 850 2518; fax: + 81 29 850 2518. E-mail address: [email protected] (H. Fujimaki). 0892-0362/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ntt.2011.07.005
range of nervous system cell types, such as microglia (Glezer et al., 2007; Olson and Miller, 2004), astrocytes (Bsibsi et al., 2006; Farina et al., 2005), oligodendrocytes (Bsibsi et al., 2002), and neurons (Lafon et al., 2006). TLR4 is crucial for the identification of lipopolysaccharides (LPS) present in the cell wall component of Gram-negative bacteria (Hoshino et al., 1999; Poltorak et al., 1998). The binding of LPS to its cognate TLR4 triggers the NF-κB transduction pathway, leading to the transcriptional activation of proinflammatory cytokines, chemokines and other genes essential for pathogen elimination (Akira et al., 2001; Beutler, 2002). TLRs might not only serve to sense microbial pathogen-associated molecular patterns (PAMPs), but also to sense certain endogenous factors that act as damage-associated molecular pattern (DAMP) molecules (Rubartelli and Lotze, 2007). Potential DAMP molecules include heat shock proteins, fibrinogen, fibronectin, heparin sulfate, soluble hyaluronan, oxidized LDL, gangliosides, fatty acids and other cues of dying cells, and these molecules can stimulate TLR4 and TLR2 signaling (Marshak-Rothstein, 2006; Beg, 2002; McMahon et al., 2005). In a brain cell injury model, the activation of TLR4 and NF-κB may be neuroprotective, either increasing cell resistance or removing toxic molecules via an increase in the phagocytic capacity of activated microglia (Glezer et al., 2006). However, little is known about the role of TLR4 in neurogenesis following environmental exposure to toxic chemicals. Previously, we reported that NMDA receptor subunit and related protein kinase and transcription factor may act as neurological biomarkers in toluene-induced developmental-phase specific neurotoxicity (Win-Shwe et al., 2010b). Moreover, we demonstrated that neurotrophins and proinflammatory cytokines are important
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neuroinflammatory mediators via TLR4-dependent NF-κB pathway in toluene-induced developmental-phase specific immunotoxicity (Win-Shwe et al., 2011). From these two studies, we suggest that a late postnatal period of mouse which corresponds with the third trimester in human may be the susceptible/critical window for toluene exposure in developmental toxicity. Our previous studies prompted us to investigate the sensitive biomarkers in toluene-induced neurotoxicity in adult mice. Thus, in the present study, we hypothesized that toluene exposure may induce neuronal damage and release DAMP molecules from damaged neurons in the mouse hippocampus, resulting the activation of TLR4 signaling pathways. These molecules may act as endogenous ligands that bind to TLR4, triggering the production of proinflammatory cytokines via the activation of NF-κB or other transcription factors and inducing neuroinflammation and neurodegeneration. In the presence of TLR4, neurons and glia cells are activated and take part in a compensatory brain repair mechanism. Therefore, in the present study, using wild type and TLR4 defective mice, we investigated the possible involvement of hippocampal TLR4 in neurotoxicity following toluene exposure. 2. Materials and methods 2.1. Animals Eight-week-old male C3H/HeN and C3H/HeJ mice were obtained from Charles River Japan Inc. (Yokohama, Japan) and were used at 10 weeks of age. Food and water were given ad libitum. This study was approved by the Ethics Committee for Experimental Animals of the University of Occupational and Environmental Health, Japan. 2.2. Exposure to toluene Toluene vapor was generated using an organic solvent gas generator (Shibata Scientific Technology Ltd.), diluted with clean, filtered air to achieve the desired gas concentrations, and then introduced into a stainless steel and glass chamber as described previously (Hori et al., 1999). The toluene concentration in the chamber was monitored using gas chromatography (Model 353B; GL Sciences, Tokyo, Japan). The average toluene levels (mean ± SD) for the 5, 50, or 500-ppm experiments were 4.9 ± 0.4, 50.5 ± 2.0, and 505 ± 11.8 ppm, respectively. Each group of mice (n= 9) was exposed to a filtered air control (0 ppm) or toluene for 6 h (from 10:00 to 16:00 h) per day, 5 days per week for 6 weeks. 2.3. Quantification of the hippocampal mRNA expression levels One day following the final toluene inhalation, the mice were sacrificed under deep pentobarbital anesthesia and the hippocampi were collected from each group of mice (n = 6) and frozen quickly in liquid nitrogen, then stored at − 80 °C until the total RNA was extracted. Briefly, total RNA extraction from the hippocampal samples was performed using the BioRobot EZ-1 and EZ-1 RNA tissue mini kits (Qiagen GmbH, Germany). Then, the purity of the total RNA was examined, and the quantity was estimated using the ND-1000 NanoDrop RNA Assay protocol (NanoDrop, USA), as described previously (Win-Shwe et al., 2007). Next, we performed first-strand cDNA synthesis from the total RNA using SuperScript RNase H − Reverse Transcriptase II (Invitrogen, USA), according to the manufacturer's protocol. Next we examined mRNA expression of 18S, TLR4, NF-κB activating protein (NF-κB), hyaluronan synthase 1(Has1), and heat shock protein 70 (Hspa-1 α) by real-time RT-PCR (Applied Biosystems Inc., USA). The tissue 18S rRNA level was used as an internal control. The primer sequences for 18S were forward 5′TACCACATCCAAGAAGGCAG-3′, reverse 5′-TGCCCTCCAATGGATCCTC3′ and TLR4 were forward 5′-AAGGCATGGCATGGCTTACAC-3′, reverse
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5′-GGCCAATTTTGTCTCCACAGC-3′. Some primers (NF-κB, ID_67050; Has1, ID_15116; and Hspa-1 α, ID_193740) were purchased from Qiagen, Sample & Assay Technologies (Qiagen GmbH, Germany). Then, the relative expression levels of the TLR4-related transduction pathway mRNAs were individually normalized to the 18S rRNA content in the respective samples and expressed as mRNA signals per unit of 18S rRNA expression. 2.4. Immunohistochemistry On the day after the final toluene exposure, the brains were removed from three C3H/HeN and C3H/HeJ mice from each of the control and toluene-exposed groups after the animals had been deeply anesthetized with sodium pentobarbital; the brains were then fixed with 10% formalin. The fixed brains were dehydrated using a graded series of ethanol, cleared with xylene, and embedded in paraffin. Coronal paraffin sections were cut at a thickness of 10 μm using a cryostat and were mounted on 3-aminopropyltriethoxysilanecoated glass slides. Iba1 (microglia marker) was detected immunohistochemically in the hippocampus. Briefly, deparaffinized sections were immersed in absolute ethanol followed by 10% H2O2 for 10 min each at room temperature. After rinsing in 0.01-M phosphate buffer saline, the sections were blocked with 2% normal swine serum in PBS for 30 min at room temperature and then reacted with rabbit anti-Iba1 (diluted 1:250; Wako Pure Chemical Industries, Ltd; Osaka, Japan) in PBS for 1 h at 37 °C. The sections were reacted with biotinylated donkey anti-rabbit IgG (1:300 Histofine; Nichirei Bioscience, Tokyo, Japan) in PBS for 1 h at 37 °C before and after rinsing in PBS. The sections were then incubated with peroxidase streptavidin (1:300, ABC KIT) containing PBS for 1 h at room temperature. After rinsing in PBS, Iba1 immunoreactivity was detected using a Dako DAB Plus Liquid System (DAKO). To determine the immunoreactivity of Iba1 in the hippocampus, photomicrographic digital images (150 dpi, 256 scales) of the hippocampal regions were taken using a CCD camera connected to a light microscope. 2.5. Statistical analysis All the data were expressed as the mean ± standard error (S.E.). The statistical analysis was performed using the StatMate II statistical analysis system for Microsoft Excel, Version 5.0 (Nankodo Inc., Tokyo, Japan). The dose–response data were analyzed using a one-way analysis of variance with a post-hoc analysis using the Bonferroni/ Dunn method. Then, data within each group was further analyzed using the Student's t-test. Differences were considered to be significant at P b 0.05. 3. Results We investigated the effect of various concentrations of toluene on the relative mRNA expression of TLR4 and compared the effects in wild-type TLR4-possessing C3H/HeN mice with those in TLR4-mutant C3H/HeJ mice to evaluate the involvement of TLR signaling. As shown in Fig. 1 (A), exposure to 50 ppm of toluene significantly increased the relative mRNA expression of TLR4 in C3H/HeN mice. There is no significant change observed between the control and the 500 ppm toluene-exposed groups. On the other hand, no increase in the relative mRNA expression of TLR4 was observed in C3H/HeJ mice (Fig. 1 (B)). TLR4 signaling ultimately leads to the induction of NF-κB, followed by the production of proinflammatory cytokines and the upregulation of cell surface molecules involved in the initiation of adaptive immune responses to pathogens (Olson and Miller, 2004). In C3H/HeN mice, the relative mRNA expression level of NF-κB activating protein was increased in the 50 ppm toluene-exposed group (P b 0.05, Fig. 1 (C)). However, exposure to 500 ppm toluene significantly suppressed the relative mRNA expression of NF-κB
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C Relative mRNA expression NF-κB/18S
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Fig. 1. Relative mRNA expression of TLR4 and NF-κB mRNAs in the hippocampi of C3H/HeN and C3H/HeJ mice. Relative mRNA expression of TLR4 in (A) C3H/HeN, (B) C3H/HeJ mice and NF-κB mRNA in (C) C3H/HeN, (D) C3H/HeJ mice following exposure to 0, 5, 50 or 500 ppm of toluene for 6 weeks. Each bar represents the mean ± SE (n = 6) (*P b 0.05).
Relative mRNA expression Hspa1-α α/18S
activating protein. In the C3H/HeJ mice, however, no significant changes were observed between the control and toluene-exposed groups (Fig. 1 (D)). Our results suggested an absence of NF-κB activation in TLR4-defective mice. Hyaluronan and heat shock protein are potential DAMP molecules or endogenous ligands for TLR4. To detect the role of TLR4 in tolueneinduced neuroinflammation, we examined the relative mRNA expression level of hyaluronan (Has1) and heat shock protein 70 (Hspa1-α) in the hippocampi of C3H/HeN and C3H/HeJ mice. The expression of Has1 mRNA was not detected in either the C3H/HeN or the C3H/HeJ mice (data not shown). In the C3H/HeN mice, the relative mRNA expression level of Hspa1-α was increased in the tolueneexposed group (P b 0.05, Fig. 2). However, in the C3H/HeJ mice, no significant difference was observed between 0 and 50 ppm toluene exposed mice. We also detected the major immune cell microglia in the hippocampi of C3H/HeN and C3H/HeJ mice after toluene exposure. We found that microglia activation was prominent in the hippocam-
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Fig. 2. Relative mRNA expression of Hspa1-α mRNA in the hippocampi of C3H/HeN and C3H/HeJ mice.Relative mRNA expression of Hapa1 in C3H/HeN and C3H/HeJ mice following exposure to 0 or 50 ppm of toluene for 6 weeks. Each bar represents the mean ± SE (n = 6) (*P b 0.05).
pal area of toluene-exposed C3H/HeN (Fig. 3 (A)) and C3H/HeJ (Fig. 3 (B)) mice, compared with corresponding control mice. Moreover, toluene-exposed C3H/HeN mice showed more prominent microglia than C3H/HeJ mice. 4. Discussion Rapidly accumulating evidence suggests that environmental toxic chemicals may cause adverse health effects by disrupting the homeostatic regulatory mechanisms of the nervous and immune systems. Toluene is a well-known neurotoxicant, and previous data from both in vivo and in vitro animal studies support the hippocampus as a target for toluene (Korbo et al., 1996; Terashi et al., 1997; Gelazonia et al., 2006). In Japan, the occupational exposure limit for toluene is 50 ppm (Japan Society for Occupational Health, 1994), while the recently updated threshold limit in the United States is 20 ppm (ACGIH, 2006). TLR4 is primarily regarded as an innate immune receptor (Takeda et al., 2003); thus, its function in the brain has been commonly attributed to neuroimmune responses. TLRs recognize patterns, rather than specific molecules, in addition to their ability to recognize physiological compounds (Ohashi et al., 2000; Okamura et al., 2001; Johnson et al., 2003). TLRs also have an innate ability to mediate a rapid response to a wide range of signals in the microenvironment, and not merely to pathogens. Both in vitro and in vivo studies have indicated that TLR4 signaling may contribute to pathogenetic changes arising from environmental exposure to toxic chemicals. A previous in vitro study stated that alveolar macrophages produce proinflammatory cytokines, such as TNF-α and IL-6, in response to airborne particulates through TLR2 and 4 (Becker et al., 2002). Moreover, the induction of airway hyperresponsiveness after ozone inhalation can reportedly occur through TLR4 (Hollingsworth et al., 2004; Garantziotis et al., 2010). In vivo studies have also shown that TLR4 is necessary for airway inflammation induced by diesel exhaust particles (Takano et al., 2002; Inoue et al., 2006). Although the role of TLR4
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Fig. 3. Microglial morphology in the hippocampus of C3H/HeN and C3H/HeJ mice.Representative digital photomicrographs of Iba1-immunostained sections taken from the hippocampal region in (A) C3H/HeN and (B) C3H/HeJ mice following exposure to toluene at 0 ppm or 50 ppm for 6 weeks (scale bar = 50 μm).
signaling in airway pathogenesis has been extensively demonstrated, the role of TLR4 in brain function after environmental toxic chemical exposure remains unclear. The present study showed that exposure to toluene may induce neuroinflammation via the TLR4–NF-κB signaling pathway, in which heat shock protein may be a potential endogenous ligand for TLR4. Instead of a dose-dependent pattern, we observed a reverse U-shaped curve for the TLR4–NF-κB induced inflammatory response in C3H/ HeN mice. No mechanism has been proposed to explain the above stimulatory effect of low levels of toluene exposure, but one possibility is that toluene inhalation contributes to neural dysfunction related to sensory stimulation, and that neurotoxic reaction and homeostatic mechanisms compensate for toxicant-induced changes (Win-Shwe and Fujimaki, 2010c). We also found that microglia activation were more prominent in toluene-exposed C3H/HeN mice than in C3H/HeJ mice. We suggest that toluene may induce DAMP molecules, such as heat shock protein, in the hippocampi of C3H/HeN mice. The induction of such molecules would, in turn, stimulate TLR4 signaling and lead to the production of proinflammatory cytokines via NF-κB-dependent or -independent pathways, resulting in neuroinflammation and leading to neuron and glia activation. Neuroprotective TLR signaling might involve both immune cells and nonimmune cells, such as glia-producing neurotrophic factors (Hanisch et al., 2008). In addition, recent evidence has indicated that TLR4 plays a role in the behavioral consequences of alcohol-induced inflammatory damage via activation of microglia and astrocyte, and induction of cytokines and inflammatory mediators in mice (Pascual et al., 2011). In conclusion, we observed different regulatory mechanisms in TLR4 signaling pathway in C3H/HeN and C3H/HeJ mice exposed to low-level toluene. Finally, our previous and present studies and other studies suggest that TLR4 and related signaling pathway may act as
potential sensitive biomarkers in neurotoxicity induced by environmental toxic chemical exposure. Conflict of interest statement All authors declare that there are no conflicts of interest. Acknowledgments This research was partly supported by a grant from the Ministry of Education, Culture, Sports, Sciences and Technology, Japan (#21390198) to H. F. We are grateful to Dr. K. Arashidani for supporting toluene exposure and Ms. K. Ohnishi, K. Taki and S. Hirayama for their technical assistance. References Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol 2001;2:675–80. American Conference of Governmental Industrial Hygienists (ACGIH). Threshold limit values and biological exposure indices guide. www.acgih.org/TLV/Studies.htm 2006. Becker S, Fenton MJ, Soukup JM. Involvement of microbial components and toll-like receptors 2 and 4 in cytokine responses to air pollution particles. Am J Respir Cell Mol Biol 2002;27:611–8. Beg A. Endogenous ligands of Toll-like receptors: implications for regulating inflammatory and immune responses. Trends Immunol 2002;23:509–12. Beutler B. Toll-like receptors: how they work and what they do. Curr Opin Hematol 2002;9:2–10. Bsibsi M, Ravid R, Gveric D, van Noort JM. Broad expression of Toll-like receptors in the human central nervous system. J Neuropathol Exp Neurol 2002;61:1013–21. Bsibsi M, Persoon-Deen C, Verwer RW, Meeuwsen S, Ravid R, Van Noort JM. Toll-like receptor 3 on adult human astrocytes triggers production of neuroprotective mediators. Glia 2006;53:688–95. Farina C, Krumbholz M, Giese T, Hartmann G, Aloisi F, Meinl E. Preferential expression and function of Toll-like receptor 3 in human astrocytes. J Neuroimmunol 2005;159:12–9.
602
T.-T. Win-Shwe et al. / Neurotoxicology and Teratology 33 (2011) 598–602
Garantziotis S, Li Z, Potts EN, Lindsey JY, Stober VP, Polosukhin VV, et al. TLR4 is necessary for hyaluronan-mediated airway hyperresponsiveness after ozone inhalation. Am J Respir Crit Care Med 2010;181:666–75. Gelazonia L, Japaridze N, Svanidze I. Pyramidal cell loss in hippocampus of young rats exposed to toluene. Georgian Med News 2006;135:126–8. Glezer I, Zekki H, Scavone C, Rivest S. Modulation of the innate immune response by NMDA receptors has neuropathological consequences. J Neurosci 2006;23: 11094–103. Glezer I, Simard AR, Rivest S. Neuroprotective role of the innate immune system by microglia. Neuroscience 2007;147:867–83. Hanisch UK, Johnson TV, Kipnis J. Toll-like receptors: roles in neuroprotection? Trends Neurosci 2008;31:176–82. Hollingsworth 2nd JW, Cook DN, Brass DM, Walker JK, Morgan DL, Foster WM, et al. The role of Toll-like receptor 4 in environmental airway injury in mice. Am J Respir Crit Care Med 2004;170:126–32. Hori H, Ishidao T, Oyabu T, Yamato H, Morimoto Y, Tanaka I. Effect of simultaneous exposure to methanol and toluene vapor on their metabolites in rats. J Occup Health 1999;41:149–53. Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, Takeda Y, et al. Cutting edge: Tolllike receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol 1999;162:3749–52. Inoue K, Takano H, Yanagisawa R, Hirano S, Ichinose T, Shimada A, et al. The role of tolllike receptor 4 in airway inflammation induced by diesel exhaust particles. Arch Toxicol 2006;80:275–9. Japan Society for Occupational Health. Toluene. Ind Med 1994;36:103–10. Johnson GB, Brunn GJ, Platt JL. Activation of mammalian Toll-like receptors by endogenous agonists. Crit Rev Immunol 2003;23:15–44. Korbo L, Ladefoged O, Lam HR, Ostergaard G, West MJ, Arlien-Søborg P. Neuronal loss in hippocampus in rats exposed to toluene. Neurotoxicology 1996;17:359–66. Lafon M, Megret F, Lafage M, Prehaud C. The innate immune facet of brain: human neurons express TLR-3 and sense viral dsRNA. J Mol Neurosci 2006;29:185–94. Marshak-Rothstein A. Toll-like receptors in systemic autoimmune disease. Nat Rev Immunol 2006;6:823–35. McMahon EJ, Bailey SL, Castenada CV, Waldner H, Miller SD. Epitope spreading initiates in the CNS in two mouse models of multiple sclerosis. Nat Med 2005;11:335–9. Ohashi K, Burkart V, Flohé S, Kolb H. Cutting edge: heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex. J Immunol 2000;164:558–61.
Okamura Y, Watari M, Jerud ES, Young DW, Ishizaka ST, Rose J, et al. The extra domain A of fibronectin activates Toll-like receptor 4. J Biol Chem 2001;276:10229–33. Olson JK, Miller SD. Microglia initiate central nervous system innate and adaptive immune responses through multiple TLRs. J Immunol 2004;173:3916–24. Pascual M, Baliño P, Alfonso-Loeches S, Aragón CM, Guerri C. Impact of TLR4 on behavioral and cognitive dysfunctions associated with alcohol-induced neuroinflammatory damage. Brain Behav Immun 2011;25(Suppl 1):S80–91. Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 1998;282: 2085–8. Rubartelli A, Lotze MT. Inside, outside, upside down: damage-associated molecularpattern molecules (DAMPs) and redox. Trends Immunol 2007;28:429–36. Takano H, Yanagisawa R, Ichinose T, Sadakane K, Yoshino S, Yoshikawa T, et al. Diesel exhaust particles enhance lung injury related to bacterial endotoxin through expression of proinflammatory cytokines, chemokines, and intercellular adhesion molecule-1. Am J Respir Crit Care Med 2002;165:1329–35. Takeda K, Kaisho T, Akira S. Toll-like receptors. Annu Rev Immunol 2003;21:335–76. Terashi H, Nagata K, Satoh Y, Hirata Y, Hatazawa J. Hippocampal hypoperfusion underlying dementia due to chronic toluene intoxication. Rinsho Shinkeigaku 1997;37:1010–3. Win-Shwe TT, Yamamoto S, Nakajima D, Furuyama A, Fukushima A, Ahmed S, et al. Modulation of neurological related allergic reaction in mice exposed to low-level toluene. Toxicol Appl Pharmacol 2007;222:17–24. Win-Shwe TT, Mitsushima D, Yamamoto S, Funabashi T, Fujimaki H. Strain differences in extracellular amino acid neurotransmitter levels in the hippocampi of major histocompatibility complex congenic mice in response to toluene exposure. Neuroimmunomodulation 2009;16:185–90. Win-Shwe TT, Tsukahara S, Yamamoto S, Fukushima A, Kunugita N, Arashidani K, et al. Up-regulation of neurotrophin-related gene expression in mouse hippocampus following low-level toluene exposure. Neurotoxicology 2010a;31:85–93. Win-Shwe TT, Yoshida Y, Kunugita N, Tsukahara S, Fujimaki H. Does early life toluene exposure alter the expression of NMDA receptor subunits and signal transduction pathway in infant mouse hippocampus? Neurotoxicology 2010b;31:647–53. Win-Shwe TT, Fujimaki H. Neurotoxicity of toluene. Toxicol Lett 2010c;198:93–9. Win-Shwe TT, Kunugita N, Yoshida Y, Nakajima D, Tsukahara S, Fujimaki H. Differential mRNA expression of neuroimmune markers in the hippocampus of infant mice following toluene exposure during brain developmental period. J Appl Toxicol 2011. doi:10.1002/jat.1643. Mar 5.