Exp Neurobiol 2018; 27(4): 309-319
Published online August 30, 2018
© The Korean Society for Brain and Neural Sciences
Eugene Bok1, Eun Ju Cho2, Eun Sook Chung2, Won-Ho Shin1*, and Byung Kwan Jin2*
1Department of Predictive Toxicology, Korea Institute of Toxicology, Daejeon 34114, Korea.
2Department of Biochemistry and Molecular Biology, School of Medicine, Kyung Hee University, Seoul 02447, Korea.
Correspondence to: *To whom correspondence should be addressed.
Won-Ho Shin, TEL: 82-42-610-8088, FAX: 82-42-610-8157
Byung Kwan Jin, TEL: 82-2-969-4563, FAX: 82-2-969-4570
The present study investigated the effects of interleukin (IL)-4 on dopamine (DA) neurons in the substantia nigra (SN)
Parkinson's disease (PD) is a common neurodegenerative disorder characterized by progressive degeneration of nigrostriatal dopamine (DA) neurons and a concomitant motor dysfunction . Although the etiology of PD is still largely unknown, accumulating evidence suggests that progression of PD is associated with neuroinflammation [2,3]. Postmortem analyses of brains from patients with PD and experimental animal models of PD indicate that reactive microglia is common features of the PD brain [4,5]. Proinflammatory cytokines released by reactive microglia contribute to degeneration of DA neurons in the substantia nigra pars compacta (SNpc) . However, how these cytokines can lead to the substantial loss of DA neurons in the SN is a major unanswered question.
Cytokines that act as a potent regulator of neuroinflammation may have dual functions in the brain; beneficial vs harmful. Interlukin (IL)-4, a well known anti-inflammatory cytokine, plays beneficial roles in human patients with sepsis  and multiple sclerosis . IL-4 was found to play a vital role in the regulation of brain cleanup and repair after stroke as an endogenous defense mechanism . In the IL-4 deficient mice, infarction volume and neurological deficit are increased after transient focal cerebral ischemia . AAV-mediated expression of IL-4 suppressed gliosis and β-amyloidosis in beta-amyloid precursor protein+presenilin-1 Tg mice, resulting in enhanced neurogenesis and improved spatial learning . We previously showed that IL-4 may regulate neuroinflammation by inducing death of CD11b+-reactive microglia and increase neuronal survival in the cerebral cortex of LPS-treated rat . On the contrary, IL-4 was expressed in CD11b+-reactive microglia and contributed to degeneration of neurons in the thrombin and amyloid beta (Aβ)-treated hippocampus
Here we report that endogenously expressed IL-4 within reactive microglia mediates degeneration of DA neurons by increasing expression of CD68+ cells and proinflammatory cytokine such as IL-1β, disruption of blood-brain barrier (BBB) and astrocytes in the SN of LPS-treated rat. Our results suggest that IL-4 could play the detrimental roles in neurodegenerative diseases such as PD in which neuroinflammation and damage of BBB and astrocytes are implicated.
All experiments were performed in accordance with approved animal protocols and guidelines established by Kyung Hee University and Korea Institute Toxicology. Animal surgery was processed as previously described [13,15] with some modifications. Sprague Dawley rats (230~280 g) were anesthetized by injection of chloral hydrate (360 mg/kg, i.p.) and positioned in a stereotaxic apparatus. And received a unilateral administration of PBS or LPS into the right SN (anteroposterior −5.2 mm, mediolateral −2.1 mm, dorsoventral −7.8 mm from bregma), according to the atlas of Paxinos and Watson (1998). All injections were made using a Hamilton syringe equipped with a 30s gauge beveled needle and attached to a syringe pump (KD Scientific, MA, USA). Infusions were made at a rate of 0.2 µl/min for LPS (5 µg/3 µl in sterile PBS; Sigma, Saint Louis, USA) and for PBS as a control. For neutralization of IL-4, some of animals received LPS with anti-murine IL-4-neutralizing antibody (IL-4NA; 1 µg/µl; R&D Systems) or nonspecific goat IgG (gIgG; 1 µg/µl; R&D Systems) as a control. After injection, the needle was left in place for an additional 5 min before slowly retracted.
Brain tissues were prepared for immunohistochemical staining as previously described with some modifications [15,16]. In brief, animals were anesthetized with chloral hydrate (360 mg/kg, intraperitoneal injection) at the indicated time points after stereotaxic surgery and transcardially perfused. Brains were frozen sectioned using a sliding microtome into 40 µm coronal sections and collected in six separate series for immunohistochemical analysis. Six to eight sections per animal were assessed by immunohistochemistry and immunofluorescence throughout the all Fig. 1, 2, 3, 4, 5. Immunohistochemistry was performed using the avidin-biotin staining technique as previously described with some modifications. Free-floating serial sections were rinsed in PBS twice for 15 min and then quenched for 5 min at room temperature (RT) in PBS containing 3% H2O2. Sections were then rinsed in PBS twice for 15 min in and blocked for 30 min at RT in PBS containing 5% normal serum (Vector Laboratories, CA, USA), 0.2% triton X-100 (Sigma) and 1% bovine serum albumin (BSA) (Sigma). Sections were then rinsed in PBS containing 0.5% BSA twice for 15 min. Next, the sections were incubated overnight with gentle shaking at RT with PBS containing 0.5% BSA and the following primary antibodies: mouse anti-neuron-specific nuclear protein (NeuN; 1:200; Merck millipore, CA, USA) for general neurons, and rabbit anti-tyrosine hydroxylase (TH; 1:2000; Pel-Freez, Brown Deer, WI) for dopaminergic neurons; mouse anti-CD11b (1:500; Serotec, Oxford, UK), which recognizes complement receptor 3; mouse anti-CD68 (1:100; Serotec), which recognizes specific for glycosylated lysosomal antigen for microglia; rabbit anti-ionized calcium binding adaptor molecule 1 (Iba1; Wako, Osaka, Japan) for microglia and macrophage, glial fibrillary acidic protein (GFAP; Sigma) for astrocyte. Sections were then rinsed in PBS containing 0.5% BSA twice for 15 min and incubated for 1 h at RT in biotin-conjugated mouse (1:400; KPL, MD, USA) or rabbit (1:400, Vector Laboratories) secondary antibody. Sections were rinsed again in PBS containing 0.5% BSA and incubated for 1 h at RT in avidin-biotin complex (Vector Laboratories). After rinsing twice in 0.1M phosphate buffer (PB), the signal was visualized by incubating sections in 0.05% 3,3' diaminobenzidine (Sigma) in 0.1M PB containing 0.003% H2O2. Sections were then rinsed in 0.1M PB, mounted on coated slides, and viewed under a bright-field microscope (Olympus Optical, Tokyo, Japan). To analyze the GFAP immunonegative (i.n.) area, CellSense Standard (version 1.16; Olympus) was used. Sections of SN region were obtained and total GFAP-i. n. area was drawn using ‘closed polygon’ under measure tool. For Nissl staining, sections of the SN tissue was mounted on coated slides, dried for 1 h at RT, stained with 0.5% cresyl violet (Sigma), dehydrated, coverslipped, and then analyzed under a bright-field microscope (Olympus Optical).
For immunofluorescence staining, tissue sections were processed as previously described with some modifications . Briefly, free-floating sections were mounted on coated slides and dried for 30 min at RT. After washing in PBS, sections were incubated in PBS containing 5% normal serum, 0.2% triton X-100 and 1% BSA for 30 min and rinsed three times with PBS containing 0.5% BSA. Then the sections were incubated in goat anti-Interleukin-4 (IL-4; 1:1000, R&D Systems, MN, USA) at RT. The next day, tissues were rinsed and incubated with Alexa Fluor 594-conjugated chicken anti-goat IgG (1:2000, Invitrogen, OR, USA). For double-immunofluorescence staining, the sections were incubated in a following primary antibodies combination. A rabbit anti-TH, a mouse anti-CD11b, a rabbit anti-Iba1, a mouse anti-CD68 (1:100) and either a goat anti-IL-4 overnight at 4℃. A goat anti-IL-1β (1:200; R&D systems, MN, USA), and either a mouse anti-CD11b. After washing in PBS containing 0.5% BSA, the sections were incubated simultaneously with a mixture of following secondary antibodies for 1 h at RT. Alexa Fluor 594-conjugated chicken anti-goat IgG (1:5000; Invitrogen) with a Alexa Fluor 488-conjugated donkey anti-mouse IgG (1:5000; Invitrogen) or anti-rabbit IgG (1:5000; Invitrogen); Alexa Fluor 488-conjugated donkey anti-mouse IgG with Alexa Fluor 594-conjugated chicken anti-goat IgG or anti-rabbit IgG. After washing slides were coverslipped with Vectashield medium (Vector Laboratories). Stained samples were analyzed under confocal laser-scanning microscope (Carl Zeiss, Germany). To analyze the localization of different antigens in double-stained samples, images were obtained from the same area and merged using interactive software. Imaging data were analyzed in Image J (National Institutes of Health). ImageJ with co-localization plugin was used to quantify immunofluorescence and with colour deconvolution plugin was used to quantify chromogenic signal intensity on image.
As previously described [17,18], the total number of TH+ neurons were counted in the each groups using the optical fractionator method performed on a brightfield microscope (Olympus Optical, BX51) using Stereo Investigator software (MBF Bioscience). This unbiased stereological method of cell counting is not affected by either the reference volume (SNpc) or the size of the counted elements (neurons).
Tissues were prepared as previously described  with some modifications. FITC-labeled albumin (Sigma) assay was performed for visualization of BBB leakage. After LPS or PBS injections, rats were transcardially perfused with Hank's Balanced Salt Solution containing heparin (10 U/ml) and then immediately by 10 ml FITC-labeled albumin (5 mg/ml, in 0.1 M PBS buffer) injected at a rate of 1.5 ml/min. Brains were dissected from the skull and postfixed overnight in buffered 4% PFA at 4℃. After fixation, the brains were cut into 40 µm slices using a sliding microtome. Sections were mounted on silane-coated slides, and the FITC-labeled albumin contained vessels that were examined by confocal microscopy (Carl Zeiss). To determine the total area for FITC-labeled albumin leakage, 3 images of SN region were obtained, thresholded using Image J, quantified, and normalized by value of PBS injected SN.
Brain tissues from the ipsilateral SN were dissected at one day after LPS or PBS injection with IL-4NA or gIgG, and total RNA was extracted in a single step using RNAzol B (Tel-Test, Friendswood, TX) following the instructions of the manufacturer. Total RNA was reverse transcribed into cDNA using AMV reverse transcriptase (Promega, WI, USA) and random primers (Promega). The primer sequences used in this study were as follows: 5′-TGATGTTCCCATTAGACAGC-3′ (forward) and 5′-GAGGTGCTGATGTACCAGTT-3′ (reverse) for IL-1β (378 bp); and 5′-TCCCTCAAGATTGTCAGCAA-3′ (forward) and 5′-AGATCCACAACGGATACATT-3′ (reverse) for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 308 bp). The PCR amplification consisted of 30 cycles of denaturation at 94℃ for 30 s, annealing at 50℃ for 30 s and extension at 72℃ for 30 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, Tokyo, Japan).
The production of IL-1β from rat SN was determined by enzyme-linked immunosorbent assay (ELISA) techniques. Tissues were homogenized in 100 µl ice-cold RIPA buffer (60 mM NaCl, 0.1% sodium dodecyl sulfate, 1% Nonidet P-40, 0.5% sodium deoxycholic acid, 50 mM Tris, pH 8.0) and centrifuged at 14,000 g at 4℃ for 15 min. Equal amounts of protein (25 µg) from each sample were placed in ELISA kit strips coated with the appropriate antibody. ELISA was then performed according to the manufacturer's instructions (Thermo Fisher Scientific, Fremont, CA).
All values are expressed as the mean±SEM. Statistical significance (p<0.05 for all analyses) was assessed by ANOVA using Instat 5.01 (GraphPad Software, San Diego, CA, USA).
LPS (5 µg) or PBS as a control were unilaterally injected into the rat SN. 7 d later, brain sections were processed for immunostaining for TH to detect DA neurons and NeuN to detect general neurons, and Nissl staining in the SN. In the LPS-treated SN, the significant loss of TH+ cells (Fig. 1E, F), NeuN+ cells (Fig. 1G), and Nissl+ cells (Fig. 1H) were observed, compared to the PBS-treated SN (Fig. 1A~D), respectively. When quantified by stereological counting, LPS decreased the number of TH+ and Nissl+ cells in the SN by 62% and 73%, respectively, compared to PBS control (Fig. 1I).
To determine microglial activation, sections were immunostained with the antibodies against CD11b, ionized calcium binding adaptor molecule 1 (Iba1) and CD68 as described . In the PBS-injected SN, CD11b+ (Fig. 1J, K) and Iba1+ (Fig. 1L, M) cells exhibited the typical ramified morphology of resting microglia. In the LPS-lesioned SN, the majority of CD11b+ (Fig. 1P, Q) and Iba1+ (Fig. 1R, S) cells displayed an activated morphology, including larger cell bodies with short, thick, or no processes, where DA neurons were degenerated. CD68+ phagocytic cells were detected in the LPS-lesioned SN (Fig. 1T, U), whereas few of CD68+ cells were detected in the PBS-treated SN (Fig. 1N, O).
As we previously showed LPS-induced IL-4 expression in the rat cerebral cortex , we investigated if LPS could induce IL-4 expression in the LPS-lesioned rat SN. After unilateral injection of LPS or PBS as a control, brain sections were processed for immunostaining for IL-4. In the PBS-injected SN, IL-4+ cells were undetectable (Fig. 2A). In the LPS-lesioned SN, substantial number of IL-4+ cells were observed as early as 1 d post-LPS (Fig. 2B), dramatically increased at 3 d post-LPS (Fig. 2C), which was detectable at 7 d post-LPS (Fig. 2D).
To verify the cell type, which is expressing IL-4 in the SN, brain sections were processed for immunostaining with antibodies against IL-4 and TH (Fig. 2E, I), IL-4 and CD11b (Fig. 2F, J), IL-4 and Iba1 (Fig. 3G, K), or IL-4 and CD68 (Fig. 3H, L) at 3 d after intranigral injection of PBS as a control (Figs. 2E~H) or LPS (Fig. 2I~L) and both images were merged. The levels of IL-4 expression were significantly increased within CD11b+ (Fig. 2J, M), Iba1+ (Fig. 2K, M), and CD68+ (Fig. 2L, M) cells in the LPS-treated SN, compared to the PBS-treated SN (Fig. 2F~H, M). Expression of IL-4 were undetectable in TH+ neurons in the both LPS and PBS-treated SN (Fig. 2E, I, M). These results indicate that IL-4 expression was localized exclusively within activated microglia.
To investigate the potential function of IL-4 in the LPS-lesioned rat SN, IL-4NA or gIgG as a control were co-injected with LPS into the SN. Pharmacological inhibition of IL-4 function by IL-4NA significantly attenuated LPS-induced neurotoxicity, which was demonstrated by increasing the number of TH+ (Fig. 3E, F, I) and Nissl+ (Fig. 3H, I) cells in the SN, compared to the gIgG-treated control (Fig. 3A, B, D, I). Additional NeuN staining demonstrated the significant increase in NeuN+ cells in IL-4NA-treated LPS-lesioned SN (Fig. 3G), compared with the gIgG-treated LPS-lesioned SN (Fig. 3C).
To determine the IL-4 actions on microglia, sections were immunostained with antibodies against CD11b, Iba1 and CD68. IL-4NA-treated LPS-lesioned SN exhibited the significant inhibition of microglial activation as determined by immunohistochemical staining of CD11b+ (Fig. 3P, Q), Iba-1+ (Fig. 3R, S) and CD68+ (Fig. 3T, U) cells when compared with gIgG-treated LPS-lesioned SN as a control (Fig. 3J~O), respectively.
As IL-4 seemed to induce microglial activation, we investigated if it could exert neurotoxicity by regulating the expression of IL-1β within activated microglia. Immunohistochemical staining showed that IL-4NA treatment significantly decreased the number of CD68+ cells in the SN by 43% at 1d post injection (Fig. 4A, B). Additional immunohistochemical analysis demonstrated a colocalization of IL-1β within CD11b+ cells in the SN at 1 d after LPS (Fig. 4C). The mRNA levels of IL-1β were further confirmed by RT-PCR analysis. The results demonstrated that IL-4NA treatment significantly attenuated the mRNA expression of IL-1β by 56% in the LPS-lesioned SN, compared with the gIgG-treated LPS-lesioned SN (Fig. 4D, E). Similar to RT-PCR data, ELISA analysis demonstrated that LPS enhanced the expression of IL-1β protein in the SN at 1 d after LPS compared with the PBS control. In the IL-4NA-treated LPS-lesioned SN, the amount of IL-1β were significantly decreased compared with the LPS-lesioned SN (Fig. 4F), indicating that IL-4 regulates IL-1β expression in the LPS-lesioned SN
As IL-1β produced by microglia can alter BBB permeability
As astrocytes are closely associated with maintenance and formation of BBB , the effects of IL-4 on astrocyte were further investigated. SN tissues adjacent to those used to BBB leakage analysis in Fig. 5A~E were immunostained with antibody against GFAP to detect astrocytes. At 3 d post-PBS, the GFAP+ cells displayed ramified morphology as a resting astrocytes in the SN (Fig. 5F). In contrast, area lacking of GFAP+ cells [GFAP(−) area] was observed as early as at 12 h and gradually and significantly increased up to at 3 d in the LPS-lesioned SN (Fig. 5G~J). Pharmacological inhibition of IL-4 function by IL-4NA almost completely reduced the GFAP(−) area in the SN at 3 d post-LPS (Fig. 5E, K), compared with the LPS-lesioned SN (Fig 5D, K) or PBS control SN (Fig. 5F, K).
The present study is the first demonstration that the detrimental role of IL-4 on degeneration of DA neurons in the LPS-treated SN are associated with reactive microglia, upregulation of proinflammatory cytokines and disruption of BBB and astrocytes.
Reactive microglia found in brain of patients with PD , exacerbates disease progression and loss of DA neurons [25,26]. In our previous reports, CD11b+-reactive microglia was inhibited by capsaicin in the SN of 1-methy-4-phenylpyridinium (MPP+)-treated rat, resulting in survival of DA neurons . Inhibition of Mac-1+-, Iba-1+-, and CD68+-reactive microglia by ethyl pyruvate  and inhibition of CD68+-reactive microglia by cannabinoid receptor agonists  resulted in DA neuron survival in the SN of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. In case of LPS-treated rat SN, fluoxetine prevents the degeneration of DA neurons by inhibiting expression of Mac-1+- and CD68+-reactive microglia . The present study demonstrates that LPS significantly upregulated the expression of IL-4 and CD11b+-, Iba-1+- and CD68+-reactive microglia in the SN. In contrast, IL-4NA dramatically decreased expression of CD11b+-, Iba-1+- and CD68+-reactive microglia and the number of CD68+ cells and rescued DA neurons. These data support the hypothesis that IL-4 has the capacity to induce microglial activation, resulting in the degeneration of DA neurons in the SN of LPS-treated rat.
Astrocytes are the most abundant glial cells in the brain, provide structural and metabolic support  and produce various neurotrophic molecules, which is essential for the development and survival of DA neurons . Astrocytes are well known to maintain the BBB integrity , that has shown to be disrupted in patients with PD . BBB protects the brain from potential neurotoxic molecules in circulation via tightly regulating ion balance, nutrient transport, the entry of plasma components and blood cells . Increasing evidence shows the BBB disruption in patients with PD [33,34] and animal models of PD [35,36,37,38]. Many studies including ours showed that BBB damage contributes to the degeneration of DA neurons in the SN of MPTP-treated mice [19,20,29] and LPS-treated rats [39,40]. The present data demonstrate that LPS increases the diffusion of FITC-labeled albumin into the SN from multiple blood vessels and damage of GFAP+ astrocytes. The pharmacological inhibition of IL-4 by IL-4NA mitigates the LPS-induced disruption of BBB and astrocytes and rescues DA neurons (Fig. 5), suggesting that IL-4 induces disruption of BBB and astrocytes, resulting in degeneration of DA neuros in the SN
IL-1β is significantly upregulated in the cerebrospinal fluid of patients with PD . Accumulating evidence from several studies including ours have demonstrated that IL-1β closely associated with degeneration of DA neurons and BBB disruption in the MPTP [29,42] and LPS model of PD [35,43]. In the 6-hydroxydopamine-treated rat, a subtoxic dose of LPS increased secretion of IL-1β which may contribute degeneration of DA neuron in the SN and motor symptoms
The current results are incompatible to our previous report that IL-4 enhances neuronal survival after traumatic spinal cord injury and in LPS-treated cortex
IL-4 and IL-13 partially share their receptors that heterodimerize the IL-4 receptor alpha chain (IL-4Rα) with IL-13Rα1 forming a complex capable of binding IL-4 or IL-13 [49,50]. IL-13Ra1 is expressed in DA neurons and contributes to oxidative stress and lack of IL-13Rα1 delays the loss of DA neurons during restraint stress and LPS-induced acute stress in mice [51,52]. Although the present study did not provide the effect of IL-4 which could be specific and relevant to the pathogenesis of PD, further study involving expression of IL-4R and/or IL-13R on DA neurons could be useful to validate if IL-4 effects can be specific and relevant to the pathogenesis of PD.
Finally, neuroinflammation can be produced by reactive microglia, proinflammatory cytokines, disruption of BBB and astrocytes, which induces onset and/or progression of degeneration of DA neurons occurring in PD. Therefore, it is likely that endogenously expressed IL-4 might serve as the neurotoxic molecule by enhancing PD-related inflammatory processes in the LPS-treated rat model of PD.