IL-13-Induced Oxidative Stress via Microglial NADPH Oxidase Contributes to Death of Hippocampal Neurons In Vivo¹

Microglia are major inflammatory cells in the CNS that are activated in response to brain injury (1, 2). As activated microglia gain neurotoxic functions through the production of neurotoxic and inflammatory mediators (1, 3), brain inflammation is closely associated with the pathogenesis of various neurodegenerative diseases, such as Alzheimer’s disease (AD)³ (4, 5). Several lines of evidence confirm microglial activation in the brains of AD patients (6) as well as in hippocampi of AD animal models following β-amyloid (Aβ) administration (7, 8).

Activated microglia produce reactive oxygen species (ROS), such as superoxide (O₂⁻) and O₂⁻-derived oxidants, which induce or exacerbate neurotoxicity by inflicting oxidative stress on cortical (9) and dopaminergic (10, 11), as well as spinal motor neurons (12). Previous studies additionally show that microglia-derived NADPH oxidase stimulates ROS production. NADPH oxidase is a multisubunit enzyme composed of cytosolic (gp40^phox, p47^phox, p67^phox, and GTP-binding protein P21-Rac1) and plasma membrane (gp91^phox and gp22^phox) subunits that catalyze the generation of superoxide radicals from oxygen (13). Activation of NADPH oxidase leads to translocation of cytosolic proteins to the plasma membrane where they assemble with other membrane-associated proteins for activity.

Thrombin activates the NADPH oxidase complex in microglia and neurons, which in turn stimulates ROS production and oxidative stress, leading to death of hippocampal neurons in vivo and in vitro (14, 15). The brains of AD patients exhibit symptoms of oxidative stress, including oxidative modifications to proteins (16), lipids (17), and DNA (18). These findings collectively suggest that NADPH oxidase-mediated oxidative stress contributes to neurodegeneration in AD (5). This hypothesis is strongly supported by the finding that NADPH oxidase is activated in AD brains, resulting in ROS formation (19).

There is growing evidence suggesting that microvascular damage is involved in the neuropathogenesis of AD (20, 21). AD patients may have increased blood-brain barrier permeability, resulting in increased serum protein accumulation within the brain extracellular space, suggesting that certain blood-derived factors are capable of inducing various pathophysiological responses (22). Among blood-derived factors, thrombin is elevated, whereas the activity of the thrombin inhibitor protease nexin I is significantly reduced in AD brains (23, 24). In addition, thrombin induces cell death in hippocampal neurons (25, 26). Collectively, these observations indicate that thrombin may act as an endogenous neurotoxin, leading to hippocampal neurodegeneration that occurs in neurodegenerative diseases such as AD.

IL-13, a well-known anti-inflammatory cytokine, suppresses the production of inflammatory mediators, such as TNF-α, IL-1β, and IL-6 from activated microglia in vivo and in vitro (27, 28, 29, 30, 31). These results were obtained after the exogenous application of IL-13 as there is no evidence of endogenous IL-13 in microglia. However, our previous studies disclose endogenous IL-13 expression in activated microglia of LPS-treated rat cerebral cortices in vivo (32). Whereas several studies have reported beneficial effects of IL-13, such as increased survival of mice in an experimental model of sepsis (33), harmful effects of IL-13 are also documented. For instance, in a mouse asthma model, allergic airway inflammation is inhibited by treatment with an anti-IL-13 mAb (34, 35). Additionally, in endothelial cells, IL-13 induces ROS production via activation of NADPH oxidase (36). In the present study, we examine whether thrombin neurotoxicity is associated with endogenous IL-13 expression within activated microglia of the rat hippocampus in vivo. We further investigate whether IL-13 mediates ROS generation through activation of NADPH oxidase, consequently contributing to the degeneration of hippocampal neurons in vivo.

All surgical experiments were performed in accordance with Guidelines and Polices for Rodent Survival Surgery provided by the Animal Care Committee of the Kyung Hee University. Female Sprague Dawley rats (230–250 g) were anesthetized by injection of chloral hydrate (360 mg/kg i.p.) and positioned in a stereotaxic apparatus (David Kopf Instruments). A midline sagittal incision was made in the scalp and holes were drilled in the skull over dorsal hippocampus using the following coordinates: 3.6 mm posterior to bregma and 2.0 mm lateral to the midline for intrahippocampal injections according to the atlas of Paxinos and Watson (37). The hole of the tip was directed down to 2.6 mm beneath the surface of the brain for the hippocampus. All injections were made using a Hamilton syringe equipped with a 30S-gauge beveled needle and attached to a syringe pump (KD Scientific). Infusions were made at a rate of 0.2 μl/min for thrombin (20 U in 4 μl of sterile PBS; from bovine plasma; Sigma-Aldrich). For neutralization of IL-13, some animals received either 20 U of thrombin or coinjection of thrombin and anti-murine IL-13-neutralizing Ab (1 μg/μl; R&D Systems) (32, 33) or nonspecific goat IgG as a control (1 μg; R&D Systems). After injection, the needle was left in place for an additional 5 min before being slowly retracted. Intact (noninjected) or PBS-injected animals were used as controls. Thrombin was essentially free from other known activated factors such as LPS, determined by LPS blocker polymyxin B (38), as well as from plasminogen and plasmin (information from the manufacturer).

Animals were anesthetized with chloral hydrate (360 mg/kg i.p.) at the indicated time points after injection and transcardially perfused with saline solution containing 0.5% sodium nitrate and heparin (10 U/ml), followed by fixation with 4% paraformaldehyde dissolved in 0.1 M phosphate buffer (PB). Brains were removed from the skull, postfixed overnight at 4°C in buffered 4% paraformaldehyde, and stored at 4°C in 30% sucrose solution until they sank. Brains were frozen, sectioned using a sliding microtome into 40-μm coronal sections, and collected in six separate series. Immunohistochemistry was performed using the avidin-biotin staining technique as described previously (15, 39). Briefly, free-floating serial sections were rinsed three times for 10 min in PBS and then pretreated for 5 min at room temperature in PBS containing 1% H₂O₂. Sections were then rinsed in PBS containing 0.3% Triton X-100 and 0.5% BSA and then preincubated for 1 h at room temperature in PBS containing 0.5% BSA. Next, the sections were incubated overnight with gentle shaking at room temperature with PBS containing 0.5% BSA and the following monoclonal primary Abs: OX-42 (1/200; Serotec), which recognizes complement receptor 3, and the neuronal-specific nuclear protein NeuN (1/400; Chemicon International) as a general stain for neurons. Sections were then rinsed in PBS and incubated for 1 h at room temperature in 1/200 biotin-conjugated anti-mouse Ab in PBS containing 0.5% BSA. Sections were rinsed again and incubated for 1 h at room temperature in avidin-biotin complex solution (Vector Laboratories). After rinsing three times in PBS, the signal was detected by incubating sections in 0.5 mg/ml 3,3′-diaminobenzidine in 0.1 M PB containing 0.003% H₂O₂. Sections were then rinsed in PBS, mounted on gelatin-coated slides, and viewed under a bright-field microscope (Olympus Optical). For Nissl staining, some of the hippocampus tissue was mounted on gelatin-coated slides, dried for 1 h at room temperature, stained with 0.5% cresyl violet (Sigma-Aldrich), dehydrated, coverslipped, and then analyzed under a bright-field microscope (Olympus Optical).

For immunofluorescence staining, sections were processed as described previously (15). Briefly, sections were mounted on gelatin-coated slides, dried for 1 h at room temperature, and washed twice in PBS. Slides were incubated for 30 min in PBS containing 0.2% Triton X-100. After blocking with 0.5% BSA, slides were incubated overnight at 4°C with Abs selective for goat anti-IL-13 Ab (1/ 2000; R&D Systems), rabbit anti-inducible NO synthase (iNOS) (1/200; Upstate Biotechnology), goat anti-TNF-α (1/100; R&D Systems), and goat anti-IL-1β (1/200; R&D Systems). After thorough rinsing in PBS, slides were covered for 1 h at room temperature with a mixture of Texas Red-conjugated anti-goat IgG (1/200; Vector Laboratories) and fluorescein-conjugated Lycopersicon esculentum (tomato) lectin (1/200; Vector Laboratories). Slides were then rinsed three times with PBS, coverslipped with Vectashield medium (Vector Laboratories), and analyzed using an IX71 confocal microscope (Olympus Optical).

Hydroethidine histochemistry was performed for in situ visualization of O₂⁻ and O₂⁻-derived oxidants (10, 15). Forty-eight hours after injection, hydroethidine (1 mg/ml in PBS containing 1% DMSO; Molecular Probes) was administered i.p. After 15 min, the animals were transcardially perfused with a saline solution containing 0.5% sodium nitrate and heparin (10 U/ml) and then fixed with 4% paraformaldehyde in 0.1 M PB. After fixation, the brains were cut into 40-μm slices using a sliding microtome. Sections were mounted on gelatin-coated slides and the oxidized hydroethidine product, ethidium, was examined by IX71 confocal microscopy (Olympus Optical).

The production of IL-13 from rat hippocampus was determined by sandwich ELISA techniques. Tissues were homogenized in 400 μl of ice-cold radioimmunoprecipitation assay buffer (60 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholic acid, and 50 mM Tris, pH 8.0) and centrifuged at 14,000 × g at 4°C for 20 min. Equal amounts of protein (150 μg) from each sample were placed in ELISA kit strips coated with the appropriate Ab. Sandwich ELISA was then performed according to the manufacturer’s instructions (BioSource International).

Brain tissues from the ipsilateral hippocampus were dissected at the indicated time points after injection, and total RNA was extracted in a single step using RNAzol B (Tel-Test) following the instructions of the manufacturer. Total RNA was reverse transcribed into cDNA using Superscript II reverse transcriptase (Invitrogen) and random primers (Promega). The primer sequences used in this study were as follows: 5′-TGA TGT TCC CAT TAG ACA GC-3′ (forward) and 5′-GAG GTG CTG ATG TAC CAG TT-3′ (reverse) for IL-1β; 5′-GTA GCC CAC GTC GTA GCA AA-3′ (forward) and 5′-CCC TTC TCC AGC TGG GAG AC-3′ (reverse) for TNF-α; 5′-GCA GAA TGT GAC CAT CAT GG-3′ (forward) and 5′-ACA ACC TTG GTG TTG AAG GC-3′ (reverse) for iNOS; and 5′-TCCCTC AAG ATT GTC AGC AA-3′ (forward) and 5′-AGA TCC ACA ACG GAT ACA TT-3′ (reverse) for GAPDH. The PCR amplification consisted of 30 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s (for IL-1β, TNF- α, and iNOS), and extension at 72°C for 90 s. PCR products were separated by electrophoresis on 1.5% agarose gels, after which the gels were stained with ethidium bromide and photographed. For semiquantitative analyses, the photographs were scanned using the Computer Imaging Device and accompanying software (Fujifilm).

Brain tissues from the ipsilateral hippocampus were dissected and homogenized in ice-cold lysis buffer containing the following (in mM): 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 5 mM MgCl₂, 1 mM DTT, 0.1 mM PMSF, and protease inhibitor mixture (Sigma-Aldrich). The tissue homogenates were centrifuged at 4°C for 20 min at 14,000 × g, and the supernatant was transferred to a fresh tube. The extracts were frozen and kept at −80°C. For subcellular fractionation, protein extracts of both the cytosolic and plasma membrane factions were prepared from the ipsilateral hippocampus at the indicated time points after injection. Tissues were gently homogenized using a glass homogenizer in ice-cold buffer consisting of the following (in mM): 20 mM HEPES, 250 mM sucrose, 10 mM KCl, 1.5 mM MgCl₂, 2 mM EDTA, and protease inhibitor mixture (Sigma-Aldrich). Homogenates were centrifuged for 5 min at 500 × g at 4°C and supernatants were collected and centrifuged for 20 min at 13,000 × g at 4°C. The pellets were further centrifuged for 1 h at 100,000 × g at 4°C, and the resulting supernatants and pellets were designated as the cytosolic and plasma membrane fractions, respectively. Equal amounts of protein (50 μg) were mixed with loading buffer (0.125 M Tris-HCl (pH 6.8), 20% glycerol, 4% SDS, 10% 2-ME, and 0.002% bromphenol blue), boiled for 5 min, and separated by SDS-PAGE. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (Millipore) using an electrophoretic transfer system (Bio-Rad). The membranes were washed with TBS solution and then blocked for 1 h in TBS containing 5% skim milk. The membranes were then incubated overnight at 4°C with one of the following specific primary Abs: rabbit anti-iNOS (1/1000; Upstate Biotechnology), mouse anti-p67^phox (1/500; BD Biosciences), and rabbit anti-p47^phox (1/200; Santa Cruz Biotechnology). After washing, the membranes were incubated for 1 h at room temperature with secondary Abs (1/2000; Amersham Biosciences) and washed again. Finally, the blots were developed with ECL detection reagents (Amersham Biosciences). The blots were reprobed with Abs against β-actin (1/4000; Santa Cruz Biotechnology). To determine the relative degree of membrane purification, the membrane fraction was subjected to immunoblotting for calnexin, a membrane marker, using a rabbit polyclonal Ab against calnexin (1/4000; Santa Cruz Biotechnology). For semiquantitative analyses, the densities of bands on immunoblots were measured with the Computer Imaging Device and accompanying software (Fujifilm).

The extent of protein oxidation was assessed by measuring protein carbonyl levels with an OxyBlot protein oxidation detection kit (Chemicon International) according to the protocol of the manufacturer as described previously (15). Protein samples were prepared from rat brains harvested 48 h after injection. Subsequently, protein samples (15 μg) were mixed in a microcentrifuge tube with 5 μl of 12% SDS and 10 μl of 1× 2,4-dinitrophenylhydrazine (DNPH) solution. Ten microliters of 1× neutralization solution (a kit component) was added instead of the DNPH solution as the negative control. Tubes were incubated at room temperature for 15 min and then mixed with 7.5 μl of neutralization solution. Next, the samples were mixed in equal volumes of SDS sample buffer and separated by SDS-PAGE. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (Millipore). The membranes were then blocked for 1 h at room temperature in TBS containing 0.1% Tween 20 and 1% BSA. Membranes were incubated overnight at room temperature with the anti-DNPH Ab (1/150) and then incubated at room temperature for 1 h with secondary Abs (1/300). Blots were developed using ECL reagents (Amersham Biosciences). Proteins that underwent oxidative modification (i.e., carbonyl group formation) were identified as a band in the samples derivatized with DNPH. The OD of the bands was measured using the Computer Imaging Device and accompanying software (Fujifilm). Levels of protein carbonyls were quantified and expressed as the fold increase vs untreated controls.

The number of CA1 neurons was assessed at three levels of the dorsal hippocampus. Specifically, alternate sections were obtained at 3.3, 3.6, 4.16, and 4.3 mm posterior to the bregma and two regions from each level (n = 8 regions for each animal) were used to count cells in the CA1 region. The number of neurons within the CA1 layer was counted using a light microscope (Olympus Optical) at a magnification of ×400 and expressed as the number of CA1 neurons per millimeter of linear length as described previously (15). To maintain consistency across animals, a rectangular box (0.5 × 0.05 mm) was centered over the CA1 cell layer beginning 1.5 mm lateral to the midline. Only neurons with normal visible nuclei were counted. The mean number of CA1 neurons per millimeter for both hemispheres was calculated for each treatment group.

All values are expressed as mean ± SEM. Statistical significance (p < 0.05 for all analyses) was assessed by ANOVA using Instat 3.05 (GraphPad Software) followed by Student-Newman-Keuls analyses.

Thrombin (20 U/4 μl) or vehicle (PBS) control was unilaterally injected into the CA1 layer of the rat hippocampus. Seven days later, brains were removed and coronal sections were processed for NeuN and Nissl immunostaining. Neurons were labeled with an Ab against a nuclear protein (NeuN). Significant loss of NeuN-immunopositive (NeuN-ip) neurons was evident in the thrombin-treated CA1 layer of the hippocampus (Fig. 1,B) compared with the PBS-treated CA1 layer (Fig. 1,A). Thrombin-induced neurodegeneration was further confirmed by staining for Nissl using sections adjacent to those used for NeuN immunostaining. Consistent with NeuN immunostaining data, a dramatic reduction in Nissl-stained cells was evident in the CA1 area of the hippocampus in thrombin-treated animals (Fig. 1,D) compared with PBS-treated animals (Fig. 1 C).

Commercial thrombin contains impurities responsible for microglial activation (40, 41). Hirudin, a thrombin inhibitor, blocks thrombin-induced neuronal death in rat mesencephalic (39), hippocampal (supplemental Fig. 1⁴), and human fetal neurons (42) in cultures deprived of microglia and enhances the survival of hippocampal neurons after ischemia (26). The data suggest that thrombin induces neuronal death, which indirectly triggers microglial activation (11, 43, 44). Therefore, it is logical to infer that activation of microglia as a consequence of neuronal injury represents a risk factor that triggers the onset of a cascade of events leading to the ongoing neurodegeneration of hippocampal neurons in vivo. Accordingly, we examined microglial activation following neuronal degeneration in the CA1 area of the hippocampus injected with thrombin in vivo (supplemental Fig. 2). Rat brain sections were obtained 24 h after injection and immunostained with OX-42 Abs recognizing complement receptor 3. In the PBS-injected CA1 layer of the hippocampus, OX-42-immunopositive microglia exhibited the typical ramified morphology of resting microglia (Fig. 2, A and B). In contrast, in the thrombin-injected hippocampus, the majority of OX-42-immunopositive microglia displayed activated morphology, including larger cell bodies with short, thick, or no processes (Fig. 2, C and D).

Next, we investigated whether thrombin might induce IL-13 protein expression in the hippocampal CA1 layer. Immunohistochemical analysis revealed high expression of IL-13 (Fig. 2, E–H) as early as 8 h after thrombin injection, which was maintained up to 14 days, in the hippocampal CA1 layer (Fig. 2. E–H). The IL-13 level was measured after thrombin injection using the sandwich ELISA technique. Consistent with immunohistochemical evidence, IL-13 levels in the hippocampus reached a peak at 7 days and remained high for up to 14 days after thrombin. At 7 days postinjection, the IL-13 level was ∼13.8-fold (Fig. 2 I; p < 0.001) higher than that in PBS-injected (24 h postinjection) and intact (undetectable; data not shown) hippocampus controls.

To identify cells expressing IL-13 in the hippocampal CA1 layer, double immunofluorescence staining with tomato lectin (Fig. 3,A) for microglia and IL-13 Abs (Fig. 3,B), or glial fibrillary acidic protein (Fig. 3,D) for astrocytes and IL-13 Abs (Fig. 3,E), was performed at 24 h after thrombin injection. The fluorescence image from each channel was merged from the same double-labeled section (Fig. 3, C and F). These results indicate that thrombin-induced IL-13 expression is localized exclusively within activated microglia (supplemental Fig. 3).

To establish the role of IL-13 in thrombin-induced neurodegeneration in vivo, we investigated whether IL-13-neutralizing Ab (NA) might affect the neurodegenerative effects of thrombin in the hippocampal CA1 layer. NeuN immunohistochemistry (Fig. 4, A, C, and E) and Nissl staining (Fig. 4, B, D, and F) were performed 7 days after intrahippocampal coinjection of thrombin and IL-13 NA. A combination of IL-13 NA (Fig. 4, E and F) and thrombin partially protected neurons in the hippocampal CA1 layer against thrombin-induced toxicity (Fig. 4, C and D). Estimation of the percentage of NeuN-ip cells on the ipsilateral vs the contralateral side disclosed that treatment with IL-13 NA increased the number of NeuN-ip neurons in the CA1 layer of the hippocampus by ∼40% (p < 0.001; Fig. 4 G). As a control, injection of nonspecific goat IgG with thrombin had little effect, similar to that observed with injection of thrombin only.

We previously demonstrated that thrombin induces microglia-derived proinflammatory cytokines, such as TNF-α, IL-1β, and iNOS, in the hippocampus (15). Accumulating evidence (including our results) shows that activated microglia-derived proinflammatory and/or cytotoxic factors, such as TNF-α, IL-1β, and iNOS, contribute to neurodegeneration in vivo (39, 45, 46, 47). Accordingly, we examined whether thrombin-induced expression of IL-1β, TNF-α, and iNOS in the hippocampus and, consequently, neuronal survival might be affected by IL-13 NA. For this purpose, animals were killed at 8 h after intrahippocampal injection and brain tissues were dissected and prepared for RT-PCR analysis. We selected the 8-h postthrombin time point since thrombin-induced expression of TNF-α, IL-1β, and iNOS in the hippocampus was evident at this time (15). RT-PCR data showed that IL-13 NA attenuated thrombin-induced mRNA expression of TNF-α by 52% (p < 0.01), IL-1β by 60% (p < 0.01), and iNOS by 51% (p < 0.001) in the hippocampus (Fig. 5, A and B). Additional Western blot data showed that at 12 h after hrombin, IL-13 NA inhibited thrombin-induced expression of iNOS by 75% (p < 0.001) in the hippocampus (Fig. 5, C and D). To establish the cellular localization of TNF-α, IL-1β, and iNOS, brain sections were examined by double immunofluorescence staining using the appropriate Abs and tomato lectin, which is specific for microglia. TNF-α (Fig. 5,E), IL-1β (Fig. 5,F), and iNOS (Fig. 5 G) were localized in tomato lectin-positive microglia at 12 h after thrombin injection.

Previously, we demonstrated that thrombin induced ROS production through NADPH oxidase activation, and this resulted in neurodegeneration in the rat hippocampus and substantia nigra (15, 45). A number of studies have shown that IL-13 triggers ROS production via NADPH oxidase activation in endothelial cells (36, 48). Accordingly, we assessed whether IL-13 might contribute to ROS generation via activation of NADPH oxidase. Consistent with an earlier report by our group (15), in situ analysis of ROS production using hydroethidine histochemistry revealed a significant increase in the fluorescent oxidized hydroethidine products (i.e., ethidium; red fluorescence) 48 h after thrombin injection (Fig. 6,B) compared with PBS-injected controls (Fig. 6,A). In contrast, IL-13 neutralization led to a dramatic reduction in the accumulation of thrombin-induced ethidium (Fig. 6 C). The results indicate that IL-13 plays a role in ROS generation in the hippocampus in vivo.

NAPDH oxidase is a major source of ROS in microglia, particularly in the hippocampus (15). Next, we analyzed whether IL-13-induced ROS generation is mediated by activation of microglial NADPH oxidase in the thrombin-treated hippocampus in vivo. Specifically, we monitored the translocation of NADPH oxidase cytosolic subunits, p67^phox and p47^phox, from the cytosol to the plasma membrane. Western blot analyses revealed that IL-13 neutralization led to reduced levels of p67^phox and p47^phox in both the cytosolic and plasma membrane fractions compared with thrombin-injected samples (Fig. 6,D), indicating that translocation and activation of the NADPH oxidase complex is mediated by IL-13. Quantification and expression as a ratio of the membrane fraction to total protein disclosed that IL-13 NA inhibits expression of p67^phox by 62% and p47^phox by 38% (Fig. 6,E). In line with our recent findings (15), double immunofluorescence staining and confocal microscopy data showed that immunoreactivity to p67^phox (Fig. 6, F–H) and p47^phox (Fig. 6, I–K) localized to activated microglia within the CA1 layer of the hippocampus 24 h after treatment with thrombin.

To determine the extent of oxidative damage to proteins, we analyzed the protein carbonyl levels after thrombin injection, with or without IL-13 NA, into the hippocampus (15, 49). Carbonyl levels were assessed by Western blotting, and band intensities were compared. The increased protein carbonyl level in thrombin-injected samples was inhibited by 56% within 48 h after the intrahippocampal injection of IL-13 NA (Fig. 6, L and M).

Finally, we summarized the sequence of the events illustrating the role of thrombin-induced IL-13 expression in rat hippocampus in vivo (Fig. 7).

In the present study, we show that thrombin-induced neurotoxicity in the hippocampus in vivo is accompanied by IL-13 up-regulation within activated microglia. Microglia-derived IL-13 activates NADPH oxidase, resulting in ROS production and subsequent oxidative modification of proteins (supplemental Fig. 4). Further evidence for the involvement of IL-13 in oxidative stress-related neurotoxic events was obtained from data showing that an IL-13-neutralizing Ab (NA) reduced neuronal loss in the hippocampus in vivo by inhibiting NADPH oxidase activation and ROS production, thus affording eventual attenuation of oxidative damage. In addition, IL-13 mediated microglia-derived expression of proinflammatory cytokines and iNOS. The present results are the first to demonstrate that endogenous IL-13 contributes to neurodegeneration. Moreover, this neurotoxicity is mediated by oxidative stress via activation of microglia-specific NADPH oxidase, proinflammatory cytokines, and iNOS in the hippocampus.

IL-13, a well-known anti-inflammatory cytokine, suppresses the production of inflammatory mediators, including ROS, NO, IL-1β, IFN-γ, and TNF-α, from activated microglia in vivo and in vitro (27, 28, 29, 30, 31). Moreover, human rIL-13-secreting cells inhibited the development of experimental autoimmune disease in a demyelinating disease model of the CNS (28). In an experimental model of sepsis, IL-13 contributed to survival of mice by suppressing the excessive production of inflammatory cytokines and chemokines, including TNF-α and MIP-1α (33). IL-13 additionally controlled brain inflammation by reducing the iNOS and TNF-α levels in LPS-injected rat cerebral cortices in vivo, resulting in enhanced neuronal survival (32).

Notably, harmful effects of IL-13 have been reported. In a mouse asthma model, treatment with an anti-IL-13 mAb prevented progression of inflammation by inhibiting the expression of cytokines and chemokines, such as TNF-α and MIP-1α (34, 35). In addition, in endothelial cells, IL-13 induced NADPH oxidase activation and consequent production of ROS (36), possibly leading to oxidative damage and eventual cell death. The data imply that IL-13 contributes to neurotoxicity by stimulating NADPH oxidase activity for ROS production. This hypothesis is strongly supported by our current data showing that IL-13 neutralization inhibits ROS generation in thrombin-treated hippocampi by limiting oxidative damage caused by microglia-derived NADPH oxidase, eventually promoting neuronal survival.

Microglia-derived proinflammatory cytokines and cytotoxic factors are involved in thrombin-induced neurotoxicity. The levels of IL-1β (50) and TNF-α (51) are up-regulated in AD brains. Moreover, NO formation catalyzed by iNOS may be involved in the pathological processes of AD (52). Recent reports show the involvement of such factors, including TNF-α, IL-1β, and iNOS, in thrombin-induced neurodegeneration in substantia nigra (45, 53) and hippocampus (15) in vivo. Moreover, inhibition of TNF-α, IL-1β, and iNOS leads to neuronal survival. In the present study, we show that IL-13 neutralization inhibits expression of iNOS and proinflammatory cytokines after thrombin injection. Our results collectively suggest that blockade of IL-13 reduces proinflammatory cytokine expression, which results in neuronal survival in the hippocampus in vivo. Our data are strongly supported by earlier reports that treatment with an anti-IL-13 mAb inhibits expression of cytokines and chemokines, such as TNF-α and MIP-1α, in a mouse asthma model (34, 35).

It seems noteworthy that these results might be irrelevant for our previous report showing IL-13 effects on cell death of microglia, resultant neuronal survival (32). This study showed that IL-13 possibly controls brain inflammation by inducing death of activated microglia, resulting in the reduction of iNOS and TNF-α expression and eventual enhancement of neuronal survival in the LPS-treated cortex in vivo. In contrast, the current data show that IL-13 mediates the production of proinflammatory cytokines (TNF-α and IL-1β), iNOS, and NADPH oxidase-derived ROS, leading to neurodegeneration in the hippocampus in vivo. Although the underlying mechanisms are yet to be clarified, these apparent discrepancies may be attributed to the use of different stimuli (LPS vs thrombin) and/or target areas (cortex vs hippocampus). This interpretation is further supported by our unpublished observation that IL-13 expression within activated microglia was observed in both thrombin-treated cortex and LPS-treated hippocampus in a time-dependent manner. As previously reported (32), cell death of microglia was only detected in LPS-treated cortex, as evident from OX-42 immunocytochemical staining (data not shown).

To our knowledge, the present study is the first to demonstrate the possible involvement of IL-13 expressed endogenously in activated microglia after thrombin injection in the degeneration of hippocampal neurons. A number of studies, including ours, confirm the in vivo and in vitro neurotoxicity of thrombin on human fetal (42), dopaminergic (39, 45), cortical (54), and hippocampal neurons (14, 15, 26). Importantly, thrombin is increased, whereas the activity of the thrombin inhibitor protease nexin I sharply reduced in the brain of AD patients (24, 55, 56). Moreover, thrombin modulates the production of amyloid protein precursors and their cleavage into fragments detected in the amyloid plaques of AD brains (57, 58). The current findings, in combination with thrombin neurotoxicity and microglial activation, suggest that the deleterious effects of microglia-derived endogenous IL-13 are involved in oxidative stress-mediated neurodegenerative diseases, such as AD.

The authors have no financial conflict of interest.