IL-17 potentiates neuronal injury induced by oxygen–glucose deprivation and affects neuronal IL-17 receptor expression

Journal of Neuroimmunology 212 (2009) 17–25

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Journal of Neuroimmunology 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 / j n e u r o i m

IL-17 potentiates neuronal injury induced by oxygen–glucose deprivation and affects neuronal IL-17 receptor expression Dan-dan Wang a,1, Yan-feng Zhao b,1, Guang-you Wang a, Bo Sun a, Qing-fei Kong a, Kai Zhao a, Yao Zhang a, Jing-hua Wang a, Yu-mei Liu a, Li-li Mu a, De-sheng Wang b,2, Hu-lun Li a,⁎ a b

Department of Neurobiology, Harbin Medical University, Heilongjiang Provincial Key Laboratory of Neurobiology, 157 Bao Jian Road, Harbin, China, 150081 Department of Neurology, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China, 150081

a r t i c l e

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Article history: Received 29 December 2008 Received in revised form 9 April 2009 Accepted 14 April 2009 Keywords: Interlukin-17 (IL-17) IL-17 receptor (IL-17R) Oxygen-glucose deprivation (OGD) Ischemia

a b s t r a c t Interlukin-17 (IL-17) is active in a variety of brain injuries, including ischemia. The objective of this study was to test the hypothesis that IL-17 potentiates neuronal injury after stroke. Increased expression of IL-17 and IL17 receptor (IL-17R) in serum and cortex was evaluated by ELISA, RT–PCR and immunohistochemistry. In the in vitro model of oxygen–glucose deprivation (OGD), IL-17 showed a dose-dependent effect in promoting neuronal injury through IL-17-IL-17R combination which can be blocked by IL-17R/Fc chimera. Our results demonstrated the up-regulation of IL-17 and IL-17R following permanent middle cerebral artery occlusion and suggested that they contributed to stroke outcome. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Stroke is a significant cause of death and disability worldwide (Feigin et al., 2003). Disruption of glucose and oxygen supply results from occlusion of a blood vessel ultimately leading to primary neuronal losses and necrosis as well as the inflammatory responses (Barone and Feuerstein, 1999), which are characterized by the infiltration of lymphocytes into the brain, and the release of inflammatory mediators, such as pro-inflammatory cytokines (Dirnagl et al., 1999; Feuerstein et al., 1998). It was demonstrated that the inflammatory responses heavily contribute to the neuronal death following stroke (Barone and Feuerstein, 1999). Inflammation in the brain can be triggered by the expression of adhesion molecules, such as intracellular adhesion molecule-1, on the surface of capillary endothelial cells. This expression prompts the extravasation of white blood cells into the brain parenchyma, where they release multiple neurotoxic substances, such as pro-inflammatory cytokines (Danton and Dietrich, 2003). Interleukin-17 (IL-17), also referred to as IL-17A, is a prototype member of a recently identified cytokine family. IL-17 has a distinct ligand-receptor system, and is known as a proinflammatory cytokine because it prompts the release of many mediators of inflammation.

Consequently, IL-17-secreting cells have been implicated in many inflammatory diseases (Kreymborg et al., 2007; Andoh et al., 2007). In an in vitro model of the blood–brain barrier, a type of IL-17-expressed CD+ 4 T lymphocytes, called T helper 17 cells preferentially migrate across the barrier and kill human neurons by secreting granzyme B (Kebir et al., 2007). Moreover, expression of IL-17 itself could even be identified in the brain injury including Multiple sclerosis (MS) (Matusevicius et al.,1999) and stroke (Li et al., 2001; Li et al., 2005; Kostulas et al., 1999). However, less is known about the impact of IL-17 release during ischemia. There is increasing evidence that IL-17 directly impacts neuronal function (Xin et al., 2008; Viviani et al., 2007). Thus, it is necessary to explore the detailed effect of IL-17 on neuronal injury in the pathogenesis of stroke. Taken together, our work demonstrated the up-regulation of IL-17 and IL-17R in both human and mouse ischemic brain tissue, providing the hypothesis that IL-17 may cause neuronal injury during CNS inflammation after stroke. In this study, we address this question for the first time, identifying the effects of IL-17 on oxygen–glucose deprivation (OGD) hippocampal neurons by an in vitro approach and elucidate the expression of IL-17 receptor (IL-17R) before and after the induction of ischemia tress in the OGD model.

2. Materials and methods ⁎ Corresponding author. Department of Neurobiology, Harbin Medical University , 157# Baojian Road Nan gang district, Hei long jiang Harbin, China, 150081. Tel.: +86 451 87502363; fax: +86 451 86662943. E-mail addresses: [email protected] (D. Wang), [email protected] (H. Li). 1 These authors contributed equally to this work. 2 Tel.: +86 451 87502363; fax: +86 451 86662943. 0165-5728/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2009.04.007

2.1. Animals and reagents 2.1.1. Animals Adult male C57BL/6 mice weighing 25–30 g were obtained from Peking Vital River Laboratory Animal Ltd. All mice were bred and

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Fig. 1. Representative TTC-stained sections of mouse at 24 h following permanent middle cerebral artery occlusion (pMCAO).

maintained in accordance with the guidelines of the Care and Use of Laboratory Animals published by the China National Institute of Health. 2.1.2. Reagents Rabbit polyclonal anti-IL-17, anti-IL-17R and anti-glial fibrillary acidic protein antibodies were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). Mouse monoclonal anti-MAP-2 antibody was obtained from Thermo Fisher Scientific Inc. (Lafayette, CO, USA). Recombinant murine IL-17A was obtained from PeproTech EC Ltd, USA. Recombinant mouse IL-17R/Fc Chimera was from R&D Systems. RT–PCR kit and Taq enzyme were purchased from TaKaRa (Kyoto, Japan) and Roche (Hoffmann-La Roche, Inc., Switzerland) respectively. 2.2. Model of experimental murine stroke 2.2.1. Animal groups Mice were randomly divided into experimental (n = 20) and sham (n = 20) groups. Focal cerebral ischemia was induced by introduction of an intraluminal nylon thread into the right cervical internal carotid artery, as described previously (McCullough et al., 2003). This surgical manipulation is known as permanent middle cerebral artery occlusion (pMCAO). The experimental and sham groups were each divided into five subgroups, to assess different timepoints after surgical manipulation: 6 h (n = 4), 12 h (n = 4), 24 h (n = 4), 2 days (n = 4) and 6 days (n = 4). 2.2.2. Animal surgery Mice were anesthetized by intraperitoneal injection of chloral hydrate (10%). The common carotid artery was exposed and the external carotid artery was ligated. A 4–0 monofilament nylon suture with blunted tip was inserted into the internal carotid artery through an arteriectomy of the common carotid artery. The distance from the nylon thread tip to the internal carotid artery–pterygopalatine artery bifurcation was slightly less than 9 mm. The suture was then secured in place, and the animals were assessed for intraischemic neurological deficit according to the previously published description (Minematsu et al., 1992). Sham-operated mice were treated identically with the exception of insertion of the filament to produce occlusion.

5 µg of total RNA, using an RT–PCR kit from TaKaRa. PCR amplification of IL-17 and IL-17 receptors, as well as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous control, were done by Taq polymerase and the following primers: mIL-17, sense 5'-TCTCATCCAGCAAGAGATCC-3', antisense 5'-AGTTTGGGACCCCTTTACAC-3'; IL-17R, sense 5'-CCACTCTGTAGCACCCCAATG-3', antisense 5'-CCTGGAGATGTAGCCCTGGTC-3'; GAPDH, sense 5'-GCACAGTCAAGGCCGAGAAT-3', antisense 5'-GCCTTCTCCATGGTGGTGAA-3'. The numbers of amplification cycles used were 32 for GAPDH, and 36 for IL-17 and IL-17R. 2.3.3. Immunohistochemistry Immunohistochemical staining in human and mice brain tissue were compared. Paraffin-embedded human brain samples, from individuals who died of cerebral infarction were collected from the brain bank of the Department of Neurology, the First Affiliated Hospital of Harbin Medical University, China. Mouse tissue was collected from each time point of the two groups. Mice were sacrificed with an overdose of pentobarbital at all timepoints after pMCAO or sham-operation. Brain tissue samples were immediately frozen in O.C.T. and sectioned at 5 µm on a cryostat and mounted on glass slides. All incubations were carried out in humidified conditions, and slides were washed three times between steps for 5 min each in phosphatebuffered saline (PBS). Human tissue slides were first dewaxed in xylene, taken through a series of ethanols. Then, both the human and the mouse slides were fixed for 10 min in pre-cooled acetone. After washing, endogenous peroxidase was blocked by incubation in methanol containing 0.3% hydrogen peroxide for 15 min. Sections were then blocked in 5% horse serum plus 0.1% triton X-100 for 20 min at room temperature, followed by incubation overnight at 4 °C with rabbit antiIL-17 (1:100 dilution) or rabbit anti-IL-17R (1:100 dilution). After washing the primary antibody, the corresponding secondary antibody (goat anti-rabbit IgG, 1:200) was applied. Finally, DAB was used as a chromogen for visualizing labeled antigens. Nuclei were later

2.3. Detection of IL-17 and IL-17R after pMCAO 2.3.1. Measurement of serum IL-17 Measurement of IL-17 levels in mouse serum was performed with an ELISA kit specific to murine IL-17. Procedures were carried out according to the manufacturer's instructions (eBioscience Systems). Results are expressed in pg/ml values. 2.3.2. RT–PCR analysis of IL-17 and IL-17 receptors Total RNA from the right cortex of mice who suffered pMCAO for different time (24 h, 48 h, 96 h) was obtained by TRIzol extraction as recommended by Invitrogen. Reverse transcription was performed with

Fig. 2. ELISA of IL-17 secretion in the serum of operated mice after permanent middle cerebral artery occlusion (pMCAO). Serum from mice of each time point was collected and assayed for cytokine levels by ELISA. The values shown are the means ± S.D. ⁎ P b 0.05; ⁎⁎⁎P b 0.001 compared with control values (Student's t test; n = 4/group). The data represent typical samples performed in triplicate in three independent experiments.

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0.25% trypsin. The cell suspension was then passed through a 130-μm pore filter, and the filtrate was centrifuged at 1200 rpm for 10 min. Following a washing step with 10% DMEM, the cell pellet was resuspended in Neurobasal™ Medium (GIBCOBRL) containing 2% B27 Supplement (GIBCOBRL), 1 mM L-glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin. These resuspended cells were then plated in 96-well plates or on poly-L-lysine-coated glass coverslips in the 24well plates at a concentration of 5 × 104 cells/ml. Medium was renewed twice a week. All experimental treatments were performed on 10-day-old cultures, at which time they contained less than 10% astrocytes as determined by glial fibrillary acidic protein (GFAP)-immunocytochemistry. Fig. 3. Ischemia-induced IL-17 and IL-17R expression in pMCAO mice. Levels of IL-17 and IL-17R mRNA were measured with RT–PCR. IL-17 and IL-17R mRNA levels were significantly higher in ischemic cortex of pMCAO mice at 1, 2 and 4 days compared to control cortex as well as those in sham-treated mice without pMCAO.

counterstained with hematoxylin, and tissue sections were digitally imaged using Image Pro Plus software (Media Cybernetics, Silver Springs, MD). To localize IL-17+cells, double immunofluorescence was used. Frozen sections were air-dried and fixed in pre-cooled acetone. Consecutive sections were stained with rat anti-CD4 (1:100 dilution) and rabbit anti-IL-17 (1:100 dilution) antibodies overnight at 4 °C. Appropriate secondary antibodies included Cy3 anti-rabbit IgG (1:200 dilution), and FITC anti-rat IgG (1:200 dilution, both from Invitrogen) were used the next morning. As negative controls for each second layer, the same procedure as above was followed except that the primary antibodies were omitted.

2.4.2. Oxygen–glucose deprivation (OGD) treatment Oxygen–glucose deprivation (OGD) was induced in primary hippocampal cell cultures, as previously described (Xu et al., 2006), with minor modifications. Briefly, the culture medium was replaced by glucose-free Earle's balanced salt solution, and the cells were placed in an anaerobic chamber that was equilibrated for 10 min with a continuous flux of gas (95%N2/5%CO2). Hippocampal cells were incubated at 37 °C for 0.5, 1, 2, 3 and 5 h to perform the time course experiment, and for 2 h in all other experiments. Control cells were incubated in Earle's balanced salt solution with 10 mM glucose in a normoxic incubator for the same period. To terminate the oxygen– glucose deprivation, chamber was opened and the cells were returned to normoxic (95%O2/5%CO2) conditions with Neurobasal™ Medium (2% B27). 2.5. IL-17 and IL-17R/Fc chimera treatment

2.4. In vitro model for ischemic injury 2.4.1. Primary cultures of hippocampal neurons Hippocampal neuronal cultures were prepared from 17–18 d embryonic C57BL/6 mice. Briefly, hippocampi were isolated from the rest of the brain, dissociated and incubated for 15 min at 37 °C with

IL-17 was dissolved in Neurobasal™ Medium and added directly to the cell cultures at concentrations of 10, 100, 500 ng/ml for 24 h. For IL-17R neutralizing studies, a recombinant mouse IL-17R/Fc Chimera or mouse IgG isotype control (both from R&D Systems) was added to the OGD cultures for 2 h at 0.5 µg/ml, which is 40-fold the ED50 to block 10 ng/ml rmIL-17. Thereafter, cell viability was analyzed in each

Fig. 4. Immunohistochemical staining for interleukin-17 (IL-17) and interleukin-17 receptor (IL-17R) in human and mouse ischemic brain tissues. A: Increasing expression of IL-17positive cells located in mouse ischemic brain tissue. B: IL-17R labeling in mouse ischemic brain tissues. C,D: The expression of IL-17 (C) and IL-17R (D) in ischemic hemisphere of human brain. (light microscopy at a magnification of × 200).

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culture by immunofluorescence for MAP-2 and MTT assy. These experiments were replicated at least three times. Sister controls were cultured in Earle's balanced salt solution with glucose under normoxic conditions (with or without IL-17R/Fc Chimera), and then exposed to IL-17 at the same three concentrations and timepoints as the OGD cultures.

thick coronal slices (without the rostral part and caudal part) for staining with 2% 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma, St. Louis, MO, USA) in saline at 37 °C as previously described (Offner et al., 2006).

2.6. Immunocytochemistry

To determine whether there would be up-regulation of cytokine, soluble level of IL-17 was measured by ELISA in peripheral blood serum derived from C57BL/6 mice of the operation group and control group following pMCAO. As shown in Fig. 2, a gradually increased value was observed begin from 24 h after pMCAO and significant difference was shown at 24 h, 2 d and 6 d in comparison to control values in the diagram (⁎P b 0.05; ⁎⁎⁎P b 0.001.).

To evaluate cytotoxicity in neurons, coverslips from OGD and control cultures were collected and assessed by immunocytochemistry. Following fixation in 4% paraformaldehyde, neurons grown on the cover slips were exposed to mouse monoclonal anti-microtubuleassociated protein-2 (MAP)-2 (1:200 dilution). Fluorescent-labeled secondary antibodies were then applied. Immunofluorescent labeling was then analyzed under a fluorescence microscope. All MAP-2positive cells in each field (× 100 original magnification) were counted. All results were reproduced in at least three sets of experiments. To assess whether some neurons were undergoing death via apoptosis or necrosis, hippocampal cultures, in the twentyfour-well were also treated with Annexin-V fluorescein and propidium iodide (PI) according to the manufacturer's instructions (Jingmei Biotech Co. LTD).

3.2. Increased IL-17 levels in serum after pMCAO

3.3. Expression of IL-17 and IL-17R in ischemic brain tissue Increased levels of IL-17 have been observed in the serum from experimental mice. To prove more directly the pathogenetic involvement of IL-17 in ischemia conditions, cortical IL-17 and IL-17R mRNA were investigated on the ischemic side in mice at 24, 48 and 96 h after pMCAO compared to both normal cortex and sham-operated animals.

2.7. Metabolic viability To assess the effect of IL-17 administration on neuronal metabolic viability, a quantitative colorimetric MTT assay was employed. Different doses of recombinant murine IL-17 (10, 100, 500 ng/ml) were added into neuronal cultures after 2 h OGD treatment. After 24 h, MTT solution (5 mg/ml, 10 µl/well) was added, and the cultures were incubated for an additional 4 h at 37 °C in a humidified atmosphere. Formazan produced by the cells was dissolved by adding DMSO (200 µl/well) and subsequently its absorbance was measured at 490 nm with an ELISA 96-well plate reader (Bio-Rad Laboratories, Inc. USA). Results are expressed as the percentage of viable cells in OGDexposed plates, compared to control normoxic plates. 2.8. Double-immunofluorescence of IL-17R and MAP-2 Double immunofluorescent labeling for IL-17R and MAP-2 was used to identify IL-17R-positive neurons. Cultured neurons and mouse tissue sections were fixed as described above, and then incubated with a mixture of primary anti-IL-17R antibody (1:100 dilution) and antiMAP-2 antibody (1:200 dilution) overnight at 4 °C. Secondary antibodies were FITC-conjugated anti-rabbit IgG (IL-17R 1:200) and TRITC-conjugated anti-mouse IgG (MAP-2 1:200), and were incubated for 1 h at room temperature. After PBS washes, coverslips and slides were mounted with DAPI fluorescence mounting medium containing 4, 6-diamidino-2-phenylindole. For negative controls, the same procedure was followed except that the primary antibodies were omitted. 2.9. Statistical analysis Data are presented as mean ± S.D., and were analyzed using oneway ANOVA followed by the Fisher's PLSD test. A P value b0.05 was considered statistically significant. 3. Results 3.1. pMCAO pMCAO consistently induced large cerebral infarctions in the middle cerebral artery territory, the striatum and cerebral cortex (Fig. 1). Brains were harvested and coronally divided into three 2-mm-

Fig. 5. IL-17 production in CD+ 4 T lymphocytes in active areas of ischemia lesions. Double-immunofluorescence staining analyzed by fluorescence microscope. Staining for CD4 (A, green) and IL-17 (B, red) were taken from lesional cortex of pMCAO mice. C: Overlays demonstrate expression of IL-17+ CD+ 4 T cells. (light microscopy at a magnification of × 200). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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As shown in Fig. 3, in all samples IL-17 and IL-17R were present in the lesional brain, whereas no detectable bands for IL-17 mRNA or mild strip (IL-17R) were seen in nonlesional brain. Localization of IL-17+ and IL-17R+ cells was carried out by immediate immunohistochemistry staining. We found increased expression of IL-17 and its receptor in the ischemic hemisphere from both human and mice (Fig. 4), compared to the non-ischemic hemisphere. In our previous work (Li et al., 2005), astrocytes were first indicated to be an IL-17-secreting cells. As stroke is a systemic inflammation according to the brain injury, we then presumed that whether there were immunocytes infiltrating into the ischemic cortex and it is confirmed to be Th17 cells according to the double-immunofluorescence for CD4 and IL-17 (Fig. 5).

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3.4. IL-17 promotes neuronal death induced by oxygen–glucose deprivation For the above result that there was an increase expression of IL-17 and IL-17R in the ischemic hemisphere, we first determined whether IL-17 could promote neuronal death under the hypoxic microenvironment with an in vitro OGD model. Chiefly, we exposed primary hippocampus cultures to 0.5 h, 1 h, 2 h, 3 h or 5 h of OGD to assess neuronal survival. Following 24 h of return to normoxic conditions, cultures were stained for MAP-2 because the disappearance of MAP-2 immunoreactivity is associated in vivo and in vitro with neuronal injury and death (Yagita et al., 1999; Zhang et al., 2000). OGD treatment resulted in a 60% loss of neurons by 2 h (Fig. 6A), and the time 2 h was chosen for the following IL-17

Fig. 6. Neurons were vulnerable to oxygen–glucose deprivation (OGD) with an aggravation after IL-17 addition. A: Duration-dependent effect of OGD on cell viability. B: Comparison of the number of MAP-2 neurons that remain after 24 h after IL-17 addition with or without IL-17R/Fc. All results have been normalized to the number of neurons present in the OGDonly group (⁎) and IL-17R/Fc neutralization (#). C: The number of surviving neurons was assessed 24 h after OGD exposure, under different IL-17concentrations. D: MTT assay for neuronal viability to the IL-17 after OGD stress compare with sister cultures with IL-17 treatment. E: Comparison of cell viability between IL-17R/Fc pretreatment groups and IL-17 stimulation groups during OGD stress. Each bar in the figures represents the mean ± S.D. of the results from 4 wells. ⁎P b 0.01, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001, #P b 0.001.

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administration. We found that while application of IL-17 had no effect on neuronal death under 95%O2/5%CO2 culture, IL-17 consistently resulted in a significant loss of neurons following OGD stress (Fig. 6C). The number of neurons remaining in culture expressed as a percentage of those in control cultures demonstrated this marked decrease (Fig. 6B). Furthermore, this IL-17-mediated aggravation is dose-dependent (Fig. 6C) which indicated that there is a positive correlation between IL-17 dose and % neuronal loss. The different dose of IL-17-triggered neurotoxicity after OGD was markedly increased (from 35.1±2.02%, 12.2±1.24% and 6.27±1.16% respectively, virus 2 h OGD alone, to 43.05 ± 2.63%) (Fig. 6C P b 0.01). Besides MAP-2 immunoreactivity, another means to detect cytotoxicity was used. The metabolic activity of neurons was evaluated immediately 24 h after IL-17 treatment via MTT assay. There was a marked reduction in metabolic activity of cultures exposed to IL-17 compared to nontreated neurons under OGD as well as control groups (Fig. 6D). And IL-17 presents an intensified inflammation injury to neurons along a higher concentration under the oxidative stress. To determine whether IL-17R/ Fc protein protects against IL-17 induced cytotoxicity under OGD, hippocampal neurons were grown in the presence or absence of IL-17R/ Fc before exposing to IL-17. Fig. 6E shows marked protective effects of 0.5 µg/ml IL-17R/Fc on the IL-17-induced apoptosis in neurons. These data strongly suggest that membrane IL-17R is critical for IL-17 associated cell injury under OGD situation. On the other hand, the staining of membrane with FITC conjugated Annexin-V was performed to detect the translocated phospholipids phosphatidylserine, which is known to be a marker of apoptotic (Fig. 7D). Increasing number of Annexin-V and PI-positive cells was evaluated after OGD stress compared with normoxic (95%O2/5%CO2) conditions. However, there were even no differences between each concentration of IL-17 addition under OGD microenvironment (data not shown). 3.5. Neurons express IL-17R after in vivo and in vitro hypoxic stress Above, we showed that the IL-17 cytokine play an important role in OGD-induced neuronal apoptosis. To provide more compelling evidence that IL-17-mediated toxicity acts through an IL-17-IL-17R combination,

double immunofluorescent labeling was used to investigate the wakeup of IL-17R expression under oxygen–glucose deprivation stress. Previous studies have indicated the IL-17R expression in the resident neural cells but not the neurons (Ge and You, 2008). However, the transcription of IL-17R in a low-oxygen microenvironment has not been showed. Our immunolabeling revealed IL-17R-positive cells in the ischemic brain at the 1 d time point (Fig. 8A). In order to locate the IL17R, a double immunofluorescent labeling for IL-17R and MAP-2 in the ischemic brain and hippocampus cultures was performed. Fig. 8F indicated the expression of IL-17R in purified hippocampal cultures after OGD stress and IL-17R labeling was mainly somatic, with occasional positive staining in neuronal processes (Fig. 8D). In the in vivo part, after the overlays (Fig. 8C), it showed that not only neurons but also those MAP-2 negative cells indicated by the arrowheads express the receptor (Fig. 8A).

4. Discussion A growing number of recent investigations have established a critical role for cytokines in the propagation of tissue damage after ischemia. It was demonstrated that the inflammatory responses are currently being thought to contribute to the neuronal death following stroke (Barone and Feuerstein, 1999). Interleukin-17, well known for its pro-inflammatory effects (Kolls and Lindén, 2004) has been reported to play an important role in many autoimmune and inflammatory diseases (Wang et al., 2008; Ivanov et al., 2008; Kramer and Gaffen, 2007; Pongcharoen et al., 2008; Suryani and Sutton, 2007). IL-17 has been considered to be a CD+ 4 T cell-specific cytokine (Yao et al., 1995). However, there is increasing investigation reported on the IL-17 expression in the brain (Li et al., 2005; Kawanokuchi et al., 2008; Meeuwsen et al., 2003). The observation that Th17 lymphocytes transmigrate across the blood brain barrier and kill neurons by secreting granzyme B suggests that IL-17 could influence stroke-mediated pathology in the brain (Kebir et al., 2007).

Fig. 7. MAP-2 immunoreactivity indicates the extensive loss of neurons after oxygen–glucose deprivation (OGD) and IL-17 exposure. A: A low magnification micrograph of control neuronal clusters (× 100 original magnification) serves as a comparison of neuronal density for B and C. B: Neurons cultured under the OGD for 2 h show a decreased cellular density (×100). C: Few cells remain after OGD exposure and administration of recombinant murine IL-17 at a concentration of 100 ng/ml (×100). D: Annexin V-positive neurons indicative of apoptotic cells can be found in cultures exposed to OGD treatment. Original magnification is × 400.

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Fig. 8. IL-17R immunolabel in neurons of ischemic tissue or in vitro oxygen–glucose deprivation (OGD) culture. A-C: mouse brain sections after pMCAO with a low magnification (×100); D-F: neurons culture after oxidative–glucose stress (scale bar = 5 µm); C and F: Overlays show expression of IL-17R in neurons. Samples were processed for immunofluorescence staining with antibodies against IL-17R (green) and MAP-2 (red). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

In our study, up-regulation of IL-17 was evaluated in mouse serum after pMCAO (Fig. 2). However, serum levels are unlikely to completely reflect regional brain pathology. It is suggested that there may be systemic repercussions in lymphoid organs that occur in response to postischemic injury in the brain. Whether inflammation is destructive or protective may depend on how severe the ischemia is, how inflammatory cells are activated and the stage of stroke during which inflammatory responses are active. In order to explore the detailed distribution of IL-17 in the progress of cerebral ischemia, immunochemistry method and RT–PCR assay were used to detect the existence of IL-17 in the brain of human patients and ischemic mice. Levels of IL-17 were elevated in the ischemic hemispheres (Figs. 3 and 4). After immunochemistry stain, localization of IL17 positive cells were observed in ischemic hemisphere. Here raises the question: the type of the cells? In our previous study (Li et al., 2005), we have confirmed the IL-17 expressing-astrocytes which are originally polymorphous in focal ischemic brain tissue. It is reported that cytokines production by resident brain cells, including glia and neurons (Sairanen et al., 2001) as well as immunocytes are increased after a variety of injuries including stroke. In addition, after CNS injury or inflammation, astrocytes respond with hypertrophy, hyperplasia, and increased production of IL-6 and IL-1β, which are both important inducers of IL17 production (Kawanokuchi et al., 2008; Schindler et al., 1990). The up-

regulated expression of these three cytokines in the pathogenesis of mammalian central nervous system injury (Wang et al.,1995; Lehrmann et al., 1995) strongly support the possibility of multiple IL-17-expressing cells, including immunocytes. A recent study (Tzartos et al., 2008) + showed that an increased labeling for IL-17 in both CD+ 4 and CD8 T cells in MS brain lesions, as well as in astrocytes and oligodendrocytes is in uniform with Meeuwsen's report (Meeuwsen et al., 2003). Thus, we suspected that the there might be T cells. After double-immunofluorescent staining with the antibodies against mouse CD4 and IL-17, we found that there are Th17 cells infiltrating into the ischemic region (Fig. 5). This is coincident with the ability of Th17 transmigrating into the brain in MS (Kebir et al., 2007). Neurons are extremely susceptible to changes in oxygen or blood flow, and clinical studies have shown impaired neuron function after as little as 10 min of ischemia (Oechmichen and Meissner, 2006). The inflammatory responses following stroke heavily contribute to the neuronal death with the infiltrated lymphocytes and those cytokines they secreted. An in vitro study had shown the susceptibility of neurons to Th1 cell cytotoxicity (Giuliani et al., 2003). In animal models of ischemic stroke, neurons in the core rapidly become committed to die, because of changes in ion homeostasis and mitochondrial dysfunction (Beilharz et al., 1995). Thus, attention has increasingly focused on the surrounding penumbra, where neuronal death can extend for days to

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weeks. Therefore, to develop effective therapies for the longer time period, it is crucial to understand the neurotoxic mechanisms occurring in the penumbra. Our results that a coincident timing of post-ischemic injury with IL17 and IL-17R up-regulation suggests that IL-17 may participate in inflammation and neuronal survival following hypoxic stress. In the in vitro experiment, by using a defined set of neuron cultures, we describe that IL-17 is a potent effector of neuronal death. In order to assess its potential to aggravate neuronal apoptosis, different doses of IL-17 were added to the medium for 24 h after OGD treatment. An indirect method to observe neuronal injury was used by immunofluorescence detection for MAP-2 which is most applied to reflect the neuronal activity. Here we did not show the apoptotic (Annexin V-positive) or necrotic (PIpositive) cells to evaluate cytoactivity, because there were no differences between each IL-17-concentration after OGD stress, for so many injured cells before their detachment from the culture substrate. Time–course experiments showed greater neuronal death in primary hippocampal cultures exposed to after exposure to oxygen– glucose deprivation compared to control neurons (Fig. 6A). We also found that IL-17 potentiated the neural injury induced by OGD exposure. However, addition of IL-17 at concentrations up to 500 ng/ ml had no effect on neuronal apoptosis under normoxic conditions. However, when neurons were cultured with as little as 10 ng/ml IL-17 after OGD stress showed significant greater neuronal loss (approximately 65%) than those without IL-17 (Fig. 6B). The different dose of IL17-triggered neurotoxicity after OGD was markedly increased (from 35.1 ± 2.02%, 12.2 ± 1.24% and 6.27 ± 1.16% respectively, virus 2 h OGD alone, to 43.05 ± 2.63%) (Fig. 6B, P b 0.01). In the neutralization assay, neurotoxicity eventually due to IL-17 preparation was excluded by inhibiting the effect of the cytokine with IL-17R/Fc Chimera. These results demonstrate that IL-17 facilitates OGD-mediated neuronal cell death, and membrane IL-17R is critical for IL-17 associated cell injury under OGD situation. A second important finding is the change in neuronal IL-17R immunolabeling after the oxygen–glucose deprivation. Unlike the relatively restricted expression of IL-17, IL-17Rs have been found to be ubiquitously expressed in multiple cell types and tissues (Lehrmann et al., 1995; Moseley et al., 2003). However, none of these studies examined IL-17R expression associated with ischemic injury. Therefore, it is necessary for us to detect whether hypoxic stress could evoke changes in IL-17R expression. In the immunolabeling experiment of human and mice ischemic tissue, we found a large number of IL-17R positive cells in the ischemic hemispheres (Fig. 4C,3D), which is coincident with the IL-17R mRNA detection (Fig. 3) and immunofluorescent result (Fig. 8A), and after the MAP location, IL-17R was observed in the neurons of post-ischemic mouse (Fig. 8C). To a detailed investigation, primary hippocampal neurons were used to determine the IL-17R expression after 2 h OGD, with or without IL-17 addition. A qualitative result by immunofluorescence assay displayed the neuronal soma colouration, but little in the neuronal processes (Fig. 8F). Taken together, our study reveals for the first time the vulnerability of neurons to IL-17-mediated injury in the response to OGD stress is likely mediated by the IL-17 receptor. We have also shown that IL-17 can aggravate the toxic effects of brain ischemia. Our results would suggest that studying the detailed mechanism of IL-17 in neurons injury will shed more light onto the stroke pathology and might have major implications for potential therapeutic intervention. Acknowledgments This research was supported by a grant from Doctorial Innovation Foundation of Heilongjiang Province of China (No. YJSCX2008104HLJ), the Heilongjiang Provincial Substantial Technology Project (GA01C02), the Harbin Medical University Youth Science Foundation (060037) and the General Program of Technology Development Funds from the Educational Committee of Heilongjiang Province.

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