Article |
Address correspondence to Dr. Serge Rivest, Laboratory of Molecular Endocrinology, CHUL Research Center, Dept. of Anatomy and Physiology, Laval University, 2705 boul. Laurier, Quebec, Canada G1V 4G2. Tel.: (418) 654-2296. Fax: (418) 654-2761. E-mail: Serge.Rivest{at}crchul.ulaval.ca
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Abstract |
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Key Words: inflammation; lipopolysaccharide; microglia; ornithine decarboxylase; toll-like receptors
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Introduction |
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Polyamines are not only involved in growth and differentiation. In the brain, they modulate the gating of N-methyl-D-aspartate (NMDA) receptors and ion channels (Ficker et al., 1994; Lopatin et al., 1994; Fakler et al., 1995; Williams, 1997) and are involved in neurodegenerative processes (Morrison et al., 1998). Moreover, polyamines have recently been found to alter the inflammatory response in vitro. The endotoxin lipopolysaccharide (LPS) is able to increase ODC mRNA expression in monocytes (Messina et al., 1990), and spermine inhibits expression of the inducible isoform of nitric oxide synthase in LPS-treated J774.2 macrophages (Szabo et al., 1994). Intracellular polyamine levels were also found to alter macrophage-mediated cytotoxicity in vitro (Tjandrawinata et al., 1994). Furthermore, the release of pro-inflammatory cytokines by monocytes is inhibited by spermine in a spermine uptakedependent mechanism (Zhang et al., 1999). Spermine can also increase IL-10 synthesis and suppress IL-12p40 and IFN- production in LPS-stimulated macrophages (Hasko et al., 2000).
Spermine is produced at high levels in regenerating tissues and is released into the extracellular medium during cellular lysis (Clarke and Tyms, 1991). Such high levels of polyamines have previously been reported to be toxic for the brain, especially when oxidative products are generated by the interconversion pathway (for review see Seiler, 2000). Independently of the toxicity of its metabolites though, spermine can inhibit in a dose-dependent manner the release of tumor necrosis factor (TNF-
) in LPS-treated human monocytes (Zhang et al., 1997). These data indicate that at least in vitro, spermine has anti-inflammatory properties (for review see Zhang et al., 2000).
The innate immune system is characterized by an unspecific and rapid response to microbial components as various as peptidoglycan, lipoproteins, lipoteichoic acid, bacterial CpG DNA, and also the endotoxin LPS. This latter is a major glycolipid constituting the outer membrane of gram-negative bacteria. LPS is well known to activate macrophages and trigger the release of pro-inflammatory cytokines as well as arachidonic acid metabolites, and therefore is widely used as a model to trigger the innate immune system (Taveira da Silva et al., 1993). Acute endotoxemia provokes a sharp and transient induction of pro-inflammatory signaling events and transcription of genes that encode cytokines, chemokines, enzymes, proteins of the complement system, and toll-like receptor 2 (TLR2) in the central nervous system (CNS; for review see Nguyen et al., 2002). Therefore, the goal of the work was to determine whether polyamines have the ability to modulate the inflammatory reaction taking place in the brain. The data that a single systemic bolus of LPS caused a robust increase in ODC mRNA expression in numerous structures across the brain suggested a potential role of polyamines in the cascade of neuroinflammatory events. Then, we used different approaches and markers to ascertain the critical role of polyamines in the cerebral innate immunity in vivo. Furthermore, the functional role of polyamines in a model of neurodegeneration was investigated. Altogether, these data support a critical role of polyamines in the control of innate immune response in the brain, which has profound consequences on the neuronal integrity.
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Results |
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To ascertain the cellular localization of ODC in the untreated mouse brain, a multiple labeling approach was performed. As depicted by Fig. 2, the positive signal in both neuronal and microglial cells was confirmed via triple immunohistochemistry labeling and confocal laser scanning microscopy. Cytoplasmic ODC protein (Fig. 2, green) colocalized with the specific marker of neuronal nuclei NeuN (Fig. 2, blue), which provides the anatomical evidence that neurons have the ability to produce polyamines. ODC-immunoreactive cells also colocalized with a marker of microglial cells, iba1. (Fig. 2, red). However, ODC+/NeuN+ cells were clearly more numerous and widely distributed than ODC+/iba1+ cells across the cerebral tissue of mice injected systemically with the bacterial cell wall component.
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Discussion |
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ODC is narrowly regulated at the level of transcription, translation, and post-translation (Katz and Kahana, 1987; White et al., 1987; van Daalen Wetters et al., 1989; Matsufuji et al., 1995). Therefore, changes in mRNA levels may not reflect increase in ODC activity and polyamine biosynthesis in the cerebral tissue of LPS-treated mice. The presence of antizyme (Kilpelainen et al., 2000), the inhibitor of ODC, could also restrain the biosynthesis of polyamines in the brain. However, ODC activity was strongly induced in the brain of LPS-injected mice, which provides compelling evidence that this immune challenge is not only capable of triggering ODC transcription, but also putrescine biosynthesis within the cerebral tissue. Moreover, the ability of DFMO to alter both ODC activity and innate immune response to LPS indicates that polyamine biosynthesis is indeed taking place in the cerebral environment. The fact that intracerebral spermine infusion was able to exacerbate the effects of LPS adds further evidence that spermine, the most downstream polyamine from putrescine, is involved in the control of the inflammatory response in the CNS. It is interesting to note that the effects of DFMO on brain ODC activity were found only in LPS-challenged mice. Basal ODC activity is usually extremely low in the adult mouse CNS (2.83 ± 0.71 pmol CO2 per h and per mg protein; Kilpelainen et al., 2001), and ODC assay was performed with a protocol designed originally for ODC activity in the prostate, the tissue that exhibits the highest ODC activity under basal conditions (Kilpelainen et al., 2001). In the present method, ODC activity was close to the limit of quantification in the brain of control mice, but DFMO had clear effects on the enzyme activity in LPS-challenged animals. This is nonetheless not surprising because DFMO has been widely used as an ODC inhibitor, which usually leads to a complete depletion of putrescine contents as well as a significant decrease in spermidine and spermine levels (Gilad and Gilad, 2002; Hillary and Pegg, 2003).
Then, we verified whether microglial cells that are the resident macrophages of the brain were also capable of expressing ODC in response to the endotoxin. This was confirmed by multiple labeling and confocal microscopy approaches, and it is clear from these data that circulating LPS increases ODC gene expression within neurons and microglial cells. These data provide anatomical evidence that although regulation of gene encoding ODC takes place within neurons and microglia, pro-inflammatory transcripts are up-regulated essentially within microglia. These cells play a critical role in the control of the innate immune response, and are under the control of polyamines because the suicide inhibitor of ODC DFMO significantly prevented the increase in TLR2 and cytokine mRNA levels in response to circulating LPS. Although TLR2 is the receptor that recognizes gram-positive bacteria, its promoter is very sensitive to ligands that trigger nuclear factor kappa B signaling, and LPS has the ability to increase TLR2 transcription first within structures devoid of BBB and thereafter in microglial cells across the brain parenchyma (Laflamme et al., 2001; Nguyen et al., 2002). The fact that DFMO dramatically inhibited the effects of LPS on TLR2 and cytokine gene expression in all these regions indicates that decrease in putrescine levels is a profound endogenous anti-inflammatory mechanism in the brain.
The present data supporting that polyamines act as pro-inflammatory molecules are in disagreement with in vitro reports in which spermine was found to inhibit cytokine synthesis in macrophages in culture (Zhang et al., 1997, 1999, 2000). However, this discrepancy can be explain by the fact that the present experiments were performed in an in vivo model in the brain where microglial cells, the CNS resident macrophages, are in narrow paracrine relationships with other cells, such as astrocytes and neurons. Another major difference is that unlike in vivo, LPS may not be eliminated in culture, and chronic exposure to the endotoxin is known to cause tolerance of immune cells to this ligand (Nomura et al., 2000). Tolerant macrophages may behave quite differently to polyamines from parenchymal microglia that were activated in an acute manner in the present work.
The mechanism mediating the effects of circulating LPS on ODC induction across the cerebral elements remains unclear at this point. Immunohistochemistry was performed to label lipid A, which is the conserved part of LPS. The latter was not detectable in the parenchymal brain of mice that received a single i.p. bolus of LPS, and DFMO did not affect the entry of LPS across the BBB. Therefore, it seems that LPS is not directly responsible to trigger ODC activity in parenchymal elements of the brain, but this phenomenon may be attributable to intermediate factors. Indeed, production of TNF- by cells of the chp and CVOs may be a determinant endogenous mechanism involved in this process. Microglial TNF-
acts as an autocrine and paracrine factor to trigger the inflammatory reaction in the CNS during endotoxemia (Nadeau and Rivest, 2000; Nguyen et al., 2002), and this elegant mechanism is likely to explain the effects of circulating LPS on ODC in the CNS. However, we have yet to provide direct evidence supporting this cascade of events.
This issue regarding whether circulating LPS and other pathogen-associated molecular patterns have the ability to reach parenchymal elements of the brain still remains a matter of controversies. Injection with the bacterial endotoxin has been shown to cause a 36% increase in ion permeability of the BBB (Dyatlov et al., 1998) and elicit gaps between brain microvasculature endothelial cells with bulging of nuclear zones and increased numbers of vesicular Golgi complexes and ER (Persidsky et al., 1997). Lustig and colleagues have also reported that LPS can contribute to virus penetration from the blood into the CNS, a process that turns a mild viral infection into a severe lethal encephalitis caused by injury to cerebral microvasculature endothelium and modulation of BBB permeability (Lustig et al., 1992). In contrast, an opening of the BBB was observed during cerebral inflammatory responses (allergic encephalomyelitis), but not after LPS administration (de Vries et al., 1995). Despite the fact that LPS decreases endothelial resistance that may lead to enhanced transport of low and high mol wt molecules, whether the endotoxin can easily diffuse through the BBB and whether it can reach deep parenchymal brain are still open questions, and such a mechanism was not supported by the present paper.
Polyamines have the ability to modulate the immune response only in the presence of LPS, and spermine alone is unable of mimicking the effects of the endotoxin. Therefore, polyamines are not direct ligands for activating pro-inflammatory signaling and gene expression by microglial cells, at least in the present model. The brains of animals that received DFMO or spermine alone were comparable to those of vehicle-treated mice. However, polyamines seem required to transduce the secondary signal-taking place across microglial cells of LPS-treated mice. How then, do polyamines modulate the innate immune response in the CNS? They may directly alter LPS-induced signal transduction and gene expression in microglial cells (Fig. 8, model 1). Alternatively, spermine originating from neurons and microglia could exert toxic effects either by the formation of oxidative products along the interconversion pathway (Fig. 8, model 2 A) and/or by an excitotoxic processes via overactivation of NMDA receptors (Fig. 8, model 2 B). Both cases would lead to an early innate immune reaction and gene expression by microglial cells. Another possibility would be the ability of extracellular spermine to act directly on microglial cells via the polyamine transport system (Fig. 8, model 2 C) and to modulate the transcription of early genes involved in the control of the innate immune system.
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To understand the functional role of polyamines in the CNS as being neurotoxic and/or neuroprotective molecules, we used a model of neurodegeneration caused by an exaggerated inflammatory response. GCs are probably the most powerful endogenous immunosuppressors, especially for the innate immune response and the subsequent inflammatory reaction (McKay and Cidlowski, 1999). Indeed, GCs are potent inhibitors of transcription of genes encoding most of the proteins involved in the innate immune system, and a large body of evidence suggests that nuclear factor kappa B is a key step in this process (McKay and Cidlowski, 1999). Altogether, these effects of GCs ultimately lead to a decrease in pro-inflammatory signal transduction pathways and gene expression, which is an essential endogenous mechanism to avoid exaggerated responses during immunogenic challenges. As expected, the inflammatory response lasted longer in the brain of animals that received the GC receptor antagonist RU486 before the intracerebral LPS infusion (Nadeau and Rivest, 2002). Surprisingly, a single bolus of LPS caused a rapid and severe neurodegeneration only in RU486-pretreated animals (Nadeau and Rivest, 2003). This was also the case in the present work because no sign of degeneration was found in the brain of mice challenged intracerebrally with LPS and vehicle i.p., but the endotoxin became highly neurotoxic in mice treated with RU486. In this model, the stress was so intense that most of mice died within 2 h after LPS infusion; the only surviving mouse exhibited major neuronal damages at the site of LPS administration. The decrease in putrescine levels by DFMO before the RU486/LPS cotreatment was able to increase the survival rate from 7 to 66%, and was able to protect LPS-induced neurodegeneration due to inhibition of GC receptors in the brain. Therefore, polyamines are powerful neurotoxic molecules in this model of exaggerated innate immune reaction.
In conclusion, polyamines play a major role in the control of the cerebral innate immune response during microbial challenges. These data may have a major clinical impact and suggest DFMO as a potential therapeutic drug to restrain neurodegeneration in brain disorders associated with inflammation. The exact mechanisms involved with the cytotoxic effects of polyamines still need to be firmly established, but the ability of DFMO to prevent LPS-induced TNF- and the critical role of this cytokine in the LPS/RU486 model (Nadeau and Rivest, 2003) lead to this direction for future investigations.
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Materials and methods |
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Experimental protocols
The first experiment was performed to determine the expression pattern of the gene encoding ODC in the brain of mice under basal and immune-challenged conditions. To this end, mice received a single i.p. bolus of either 1 mg/kg LPS (from Escherichia coli, serotype 055:B5, lot 58H4076; Sigma-Aldrich) or vehicle solution (100 µl sterile, pyrogen-free saline), and were killed 1.5, 3, 6, and 24 h afterward as explained below. A second group of animals was pretreated with the suicide inhibitor of ODC DFMO (Ilex Oncology) for 48 h. The drug was diluted at a concentration of 2% in the drinking water, whereas the control group had access to tap water. 2 d after the beginning of the treatment, both groups were injected with either sterile saline or LPS and killed at numerous times after injection.
The third group of mice was temporarily anesthetized via an i.p. injection of a mixture of ketamine hydrochloride, xylazine, and sterile pyrogen-free saline (1.5:0.5:8.5 vol/vol/vol). The right ventricle was reached stereotaxically using the Paxinos and Franklin Atlas (David Kopf Instruments). With the incisor bar placed 2 mm below the interaural line (horizontal zero), the coordinates from bregma for the injection cannula were 0 mm anteroposterior, 1.1 mm lateral, and 2.4 mm dorsoventral. Thereafter, 100 nmol spermine (lot 126H2615; Sigma-Aldrich) or vehicle solution (pyrogen-free, sterile, distilled water) was injected with a cannula (33G; Plastics One) into the right lateral ventricle in a 1-µl volume over 2 min by means of a microinjection pump (model A-99; Razel Scientific Instruments). 5 min later, mice were injected with either LPS or vehicle and were killed at 6 h after injection time.
The fourth series of experiments consisted of determining the functional role of polyamines in a model neurodegeneration provoked by a robust innate and inflammatory response in the CNS. Mice had free access to tap water or DFMO (2% in drinking water) for a period of 2 d before the surgeries. On the day of the experiment, mice received a single i.p. administration with either the GC receptor antagonist RU486 (50 mg/kg body weight/100 µl DMSO) or vehicle (100 µl DMSO). 1 h later, mice were temporarily anesthetized via an i.p. injection of a mixture of ketamine hydrochloride, xylazine, and sterile pyrogen-free saline (1.5:0.5:8.5 vol/vol/vol). The right striatum was reached stereotaxically using the Paxinos and Franklin Atlas (David Kopf Instruments). With the incisor bar placed 2 mm below the interaural line (horizontal zero), the coordinates from bregma for the infusion cannula (33G; Plastics One) were 0 mm anteroposterior, 2 mm lateral, and 3 mm dorsoventral. Thereafter, 2.5 µg LPS (from E. coli, serotype 055:B5, lot 58H4076; Sigma-Aldrich) or vehicle solution (pyrogen-free sterile saline) was infused into the right striatum in a 1-µl volume over 2 min by means of a microinjection pump (model A-99; Razel Scientific Instruments). Animals were killed 3 or 7 d after the intracerebral infusion. Animals were anesthetized with a mixture of ketamine hydrochloride and xylazine (5:1 vol/vol) and were rapidly perfused transcardially with 0.9% saline, followed by 4% PFA in 0.1 M borax buffer (pH 9.5 at 4°C). Brains were removed and processed as described previously (Laflamme et al., 2001).
The fifth experiment was performed to verify whether the suicide inhibitor of ODC DFMO was efficient in the brain. Animals were pretreated with 2% DFMO in the drinking water for 48 h, whereas the control group had free access to tap water. 2 d after the beginning of the treatment, both groups received a single i.p. bolus with either sterile saline or LPS (1 mg/kg) and were killed 3 h later. Animals were anesthetized with a lethal mixture of ketamine hydrochloride and xylazine (5:1 vol/vol) and decapitated for a rapid removal of the brains. Cerebral hemispheres were separated and snap frozen in liquid nitrogen.
Determination of ODC activity
The cerebral hemispheres (230260 mg) were homogenized into 10 ml ice-cold homogenization buffer (50 mM Tris-HCl, pH 7.3, 50 µM pyridoxal phosphate, 1 mM EDTA, and 2.5 mM DL-dithiothreitol) with a Potter homogenizer. The homogenates were centrifuged at 25,000 g for 60 min at 4°C, and the supernatants were used for the measurement of ODC activity and protein content. ODC activity was assayed by determining the amount of 14CO2 released from L-[1-14C]ornithine as described previously (Janne and Williams-Ashman, 1971; Marchetti et al., 1988). Protein contents were determined on the cytosolic fraction by the method of Bradford (1976) using bovine gamma globulin as standard.
In situ hybridization and histological procedures
Plasmids were linearized and sense and antisense riboprobes were synthesized as described in Table I. Hybridization histochemical localization of the different transcripts was performed on every twelfth section of the whole rostrocaudal extent of each brain using 35S-labeled cRNA probes as described previously (Laflamme et al., 1999). Nissl stain was used as a general index of cellular morphology, and neuronal death was determined via the Fluoro-Jade B method (Nadeau and Rivest, 2003).
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Confocal laser scanning microscopy
Confocal laser scanning microscopy studies were performed with an oil immersion objective (X100 Plan-Apo, NA 1.35, X2 numerical zoom) and a microscope (BX-61; Olympus). CyTM2, Alexa Fluor® 546, and CyTM5 were excited sequentially at 488 nm (Ar Ion laser; Melles Griot Laser Group) set at 35% of maximum laser power, 543 nm (HeNe-G Laser; Melles Griot Laser Group) set at 25% of maximum laser power, and 647 nm (HeNe-R Laser, Melles Griot Laser Group) set at 25% of maximum laser power, respectively. Emissions from CyTM2-, Alexa Fluor® 546-, and CyTM5-labeled antibodies were recorded by photomultipliers preset respectively for EGFP (green pseudo color), TRITC (blue pseudo color), and CyTM5 (red pseudo color) fluorescent dyes in Fluoview SV 500 imaging software (Olympus). 13 1-µm confocal z-series were acquired for each area and were corrected by 2 Kahlman low speed scans. Acquired z-series images were then flattened in one image and exported in 24-bit TIFF format. For illustrating purpose, the red, green, and blue channels were subsequently separated from original picture with Adobe Photoshop® 7.0 software.
Determination of LPS within the brain parenchyma
Brain sections from the first and second groups of mice treated or not with either DFMO (2% in drinking water) or LPS i.p. (1 mg/kg body weight) were washed in KPBS and incubated for 3 h at 37°C with KPBS containing 10% goat serum. Additional tissues from animals that received a single bolus of LPS into the dorsal basal ganglia were used as positive controls. Tissues were thereafter rinsed in KPBS and incubated for 16 h at 4°C with anti-lipid A antibody (clone 43, lot HM 20463422 M11; Cell Sciences), which was diluted in sterile KPBS (1:1,000) plus 0.4% Triton X-100 and 1% BSA (fraction V; Sigma-Aldrich). Brain slices were subsequently rinsed in KPBS and incubated with a mixture of KPBS plus 0.02% Triton X-100, 1% BSA, and Alexa Fluor® 488 goat antimouse IgG Ab (1:1,500; Molecular Probes, Inc.) for 3 h at 20°C. Tissues were thereafter rinsed in KPBS. Photomicrographs were taken with the same exposure time (9 s) using a digital camera (SPOT RT Slider; Diagnostic Instruments) mounted directly on a microscope (BX-60; Olympus) equipped with an FITC-specific epifluorescence filter and connected to a Macintosh computer (Power Macintosh G4; Apple Computers).
Qualitative analysis
The relative intensity of TLR2 or TNF- mRNA signals throughout the brain of each animal was assessed on dipped emulsion slides under microscopic evaluation, and was graded according to the scale of undetectable (0), low (1), moderate (2), strong (3), or very strong (4) signal.
Quantitative analysis
Semi-quantitative analysis of the TLR2 and TNF- mRNA hybridization signal was performed on nuclear emulsiondipped slides with a CCD video camera (Sony) attached to a Bmax optical system (BX-50; Olympus) coupled to a computer. The refractory density in arbitrary unit (RDAU) of the hybridization signal was measured under darkfield illumination at a magnification of 25 using NIH Image software (version 1.61 for Macintosh PPC; Wayne Rasband, National Institutes of Health, Bethesda, MD). The RDAUs of regions of interest were then corrected for the average background signal by sampling cells immediately outside the region of interest without positive hybridization signal. Data are reported as a percentage of control (vehicle i.p., vehicle i.c.v.) of mean RDAU values (± SEM). Statistical analysis was performed by ANOVA, followed by a Bonferroni/Dunn test procedure as post-hoc comparisons with Statview software (version 4.01, Macintosh).
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Acknowledgments |
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This research was supported by a grant (FRN 12594 to S. Rivest) from the Canadian Institutes of Health Research (the former Medical Research Council of Canada, MRCC). S. Rivest is an MRCC Scientist and holds a Canadian Research Chair in Neuroimmunology.
Submitted: 23 January 2003
Revised: 4 June 2003
Accepted: 4 June 2003
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References |
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