Methamphetamine causes neurotoxicity by promoting polarization of macrophages and inflammatory response

Abstract

Macrophages, especially their activation state, are closely related to the progression of neurotoxicity. Classically activated macrophages (M1) are proinflammatory effectors, while alternatively activated macrophages (M2) exhibit anti-inflammatory properties. As a powerful addictive psychostimulant drug, coupled with its neurotoxicity, methamphetamine (Meth) abuse may lead to long-lasting abnormalities in the neuronal system. The present study investigated the effect of Meth at subtoxic concentration on macrophage activation state and its underlying toxicity to neuronal cells. PC12 and Murine RAW264.7 cells were coincubated with Meth to test its toxicity. 3-(4,5-Dimethylthiazol)-2,5-diphenyltetrazolium-bromide, enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and Western blot assays were performed to evaluate the toxicity, cytokine secretion, gene, and protein expression. Results showed that cytotoxicity was enhanced on PC12 cells after coculturing with RAW264.7 stimulated with Meth. RAW264.7 macrophages tended to switch to the M1 phenotype, releasing more nitric oxide and proinflammatory cytokines, including tumor necrosis factor α (TNFα), interleukin (IL)-12, and IL-1β, while decreasing the release of anti-inflammatory cytokine IL-10 after treatment with Meth. Meth upregulated the gene expression of IL-6, IL-1β, and TNFα and downregulated the expression of Arg-1, IL-10, and KLF4. Meth could also upregulate the protein expression of IL-1β and TNFα and downregulate the expression of Arg-1 and KLF4. However, the abovementioned effects induced by Meth were abolished by the addition of dopamine receptor D3 antagonist. In conclusion, our study demonstrated that Meth promoted macrophage polarization from M0 to M1 and enhanced inflammatory response, which provided the scientific rationale for the neurotoxicity caused by the chronic use of Meth.

Introduction

Methamphetamine (Meth) is a highly addictive, widely abused psychostimulant drug that easily crosses the blood–brain barrier and causes brain damage, leading to neuropsychiatric disorders. ^1,2 In 2012, an estimated 34 million individuals used amphetamine-type stimulants worldwide, predominantly Meth. ³ This drug may be taken orally as a pill, but the crystalline form, often referred to as “ice” or “crystal meth,” is typically smoked and may also be injected. ⁴

Acutely, Meth intake produces euphoria, hyperactivity, increased vigilance, and cardiovascular changes. However, chronic exposure or taken in binge doses, Meth can cause irreversible damage to brain cells resulting in neurological and psychiatric abnormalities. ^5,6 Studies have shown that Meth damages monoamine-containing nerve terminals in the brain and generates an imbalance in the release and reuptake of dopamine, serotonin epinephrine, and norepinephrine, resulting in dopaminergic neuronal damage and neurological abnormalities and eventually to psychiatric disorders. ^{7 –9}

Meth induces neurotoxicity via multiple mechanisms, including oxidative stress, excitotoxicity, hyperthermia, and apoptosis induction. ¹⁰ High dose of Meth caused direct neurotoxicity; however, low dose of Meth could not affect the viability of PC12 cells but resulted in neurotoxicity in animals, indicating that Meth induces neurotoxicity via other pathways. ^11,12 Neuroinflammation has been implicated as an additional mechanism, linking with several neurological disorders. ¹³ It has been well established that Meth exposure activates microglia and astrocytes in culture as well as in animal studies. ¹⁴ Such gliosis may contribute to Meth-induced neurotoxicity, as activated astrocytes and microglia produce reactive oxygen species that are harmful to neurons and proinflammatory cytokines that propagate the neuroinflammatory cascade, potentially amplifying the neuron damage. ¹⁴

Activated macrophages produce various cytokines and chemokines and exhibit diverse functions, including proinflammatory responses, tissue repair and destruction, and tumoricidal function, which is closely related to the polarized activation state. ¹⁵ This study was to explore the effect of Meth on macrophage polarization and the resultant activities on neurons.

Materials and methods

Cell line, chemicals, and reagents

PC12 and RAW264.7 cell lines were obtained from Shanghai Cell Bank (Shanghai, China). Meth was purchased from National Institutes for Food and Drug Control (Beijing, China). 3-(4,5-Dimethylthiazol)-2,5-diphenyltetrazolium-bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco BRL (Gaithersburg, MD, USA). Nitric oxide (NO), interleukin (IL)-6, IL-1β, tumor necrosis factor α (TNFα), and IL-10 assay kits were purchased from Jiancheng Institute of Biological Engineering (Nanjing, China). Cytochrome-c enzyme-linked immunosorbent assay (ELISA) kit was from R&D systems (Minneapolis, MN, USA). Cell death Detection ELISAplus kit was from Roche Applied Sciences (Switzerland). 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) were purchased from Molecular Probes (Eugene, OR, USA). Interferon γ (IFNγ), lipopolysaccharide (LPS), IL-4, and NGB2904 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Real-time polymerase chain reaction (PCR) reagents were purchased from Takara Bio Inc. (Shiga, Japan). All solvents and other chemicals used in this study were of analytical grade and purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China).

Cell culture and treatment

PC12 cell line is derived from a pheochromocytoma with extreme versatility for pharmacological manipulation and has been used to get more information about diseases of the brain. RAW264.7 is a murine macrophage-like cell line used for macrophage polarization study. PC12 cells and RAW264.7 cells were cultured in DMEM, supplemented with 10% FBS, 100 U/mL of penicillin, and 100 U/mL of streptomycin. All of the cultures were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO₂. PC12 or RAW264.7 cells were treated with Meth at different concentrations (100, 200, and 400 µmol/L). PC12 and RAW264.7 cells were cocultured using transwell^® 6-well plates (Corning^®, Tewksbury, MA, USA) to study their interaction, but PC12 and RAW264.7 cells can be collected separately. Based on the cytotoxicity assay (Figure 1), cocultured PC12 and RAW264.7 cells were treated with Meth at 400 µmol/L with or without LPS (100 ng/mL)/IFNγ (10 ng/mL) or IL-4 (10 ng/mL) for 48 h. RAW264.7 cells were treated with Meth at 400 µmol/L, supplemented with LPS (100 ng/mL)/IFNγ (10 ng/mL), or IL-4 (10 ng/mL) with or without NGB2904 (5 nM) for 48 h. ¹⁶

Figure 1.

PC12 or RAW264.7 cells were treated with methamphetamine (Meth) at the concentration of 100, 200, 400 µmol/L for 48 h. Cell viability was tested by MTT assay. Meth did not cause significant toxicity on PC12 (a) and RAW264.7 (b) cells. Samples were measured in triplicate and experiments were repeated three times. MTT: 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium-bromide.

Cell viability measurement

Cell viability was determined by MTT assay. PC12 or RAW264.7 cells or cocultured PC12 and RAW264.7 cells were treated with Meth with or without LPS (100 ng/mL)/IFNγ (10 ng/mL) or IL-4 (10 ng/mL) for 48 h. After different treatment, cells were collected and plated at a density of 3 × 10⁴/100 µL in 96-well plates. In all, 10 µL 0.5 mg/mL MTT solution was added in each well and incubated for 4 h at 37°C. Culture medium was removed, and the formazan crystals were solubilized with 100 µL dimethyl sulfoxide. The absorbance was measured at 570 nm by a microplate reader (BIO-TEK, Winooski, VT, USA). Cell viability was expressed as the percentage of the negative control, which was set to 100%. Samples were measured in triplicate, and experiments were repeated three times.

Measurement of mitochondrial membrane potential

The fluorescent probe JC-1 was used to measure the mitochondrial membrane potential (MMP). The ratio between monomer (green) and aggregate (red) fluorescence can reflect the MMP. After treatment, PC12 cells were washed with phosphate-buffered saline (PBS) and incubated with JC-1 for 15 min at 37°C in the dark. After rinsing with Hank’s solution, the fluorescence intensity of the red/green was determined on a fluorescence microplate reader (TECAN Polarion, London, UK) at an excitation of 490 nm and emission of 530 nm (green fluorescent monomers) and 590 nm (red fluorescent aggregates), respectively. The change in MMP was expressed as a percentage of the negative control, which was set to 100%. Samples were measured in triplicate, and experiments were repeated three times.

Cytochrome-c assay

The cytosolic cytochrome-c was measured by the assay kit according to the manufacturer’s instructions. After treatment, PC12 cells were washed with PBS and fractionated. After reaction, the optical density was measured by a microplate reader at 490 nm (BIO-TEK). The level of cytochrome-c was expressed as a percentage of the negative control, which was set to 100%. Samples were measured in triplicate and experiments were repeated three times.

Measurement of DNA fragmentation

Quantification of DNA fragmentation was measured by Cell Death Detection ELISAplus kit (Roche, Basel, Switzerland). After treatment, PC12 cells were washed with PBS and lyzed for 30 min. After centrifugation at 0.1 × g (Eppendorf Centrifuge 5415R, Hamburg, Germany) for 10 min at 4°C, 20 µL of supernatant was transferred to a streptavidin-coated microplate and incubated with a mixture of anti-DNA-peroxidase and anti-histone–biotin. The peroxidase amount in the immunocomplex was quantified by adding 3-ethylbenzthiazoline-6-sulfonic acid as the substrate. The absorbance of the reaction mixture was measured by a microplate reader at 405 nm (BIO-TEK). The extent of DNA fragmentation was expressed as a percentage of the negative control, which was set to 100%. Samples were measured in triplicate, and experiments were repeated three times.

Cytokine assays

After treatment, supernatant of culture medium was collected to determine the cytokine levels. NO was determined by Griess assay according to the manufacturer’s instructions. IL-6, TNFα, IL-1β, and IL-10 levels were detected using murine ELISA kits according to the manufacturer’s instructions. Samples were measured in triplicate, and experiments were repeated three times.

Quantitative real-time RT-PCR

After treatment, cells were collected and placed in TRIzol reagent to extract the total RNA. RNA samples were treated with DNase I before complementary DNA was generated by adding 1 µg of the total RNA to SuperScript master mix and performing reverse transcription. Quantitative PCR and melt-curve analyses were performed using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). Comparative C _t value method was used to quantify the expression of genes of interest in different samples. The messenger RNA (mRNA) levels were normalized to that of a housekeeping gene Rpl19 mRNA. ¹⁷ Samples were measured in triplicate, and experiments were repeated three times.

Western blot analysis

After treatment, the cells were harvested and washed with PBS. Total protein was isolated and quantified using the protein assay kit (Biyotime, Haimen, China). Cell lysates in 5× sodium dodecyl sulfate (SDS) sample buffer were boiled for 5 min, and then equal amounts of protein (40 μg) from each sample were subjected to electrophoresis on a 10% (v/v) SDS-polyacrylamide gel. After proteins were electroblotted to a polyvinylidene fluoride membrane, the membrane was blocked with PBS with Tween 20 containing 5% dried skimmed milk at room temperature, washed three times, and incubated with indicated primary antibodies at 4°C overnight, followed by incubating with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 h. After incubation, membrane was washed three times, and the antigen–antibody complexes were visualized by the enhanced chemiluminescence system (PerkinElmer, American Fork, UT, USA). Samples were measured in triplicate, and experiments were repeated three times.

Statistical analysis

Values were presented as mean ± SD and statistically analyzed by one-way analysis of variance using the Sigma Stat statistical software (SPSS Inc., Chicago, IL, USA). Differences were considered as significant at p < 0.05.

Results

Effects of meth on RAW264.7 and PC12 cell viability

Cells were treated with Meth at different concentrations. Compared to the negative control, no significant difference for the OD value was observed. PC12 and RAW264.7 cell viability were not significantly reduced after treatment with Meth, as shown by Figure 1.

Meth enhanced toxicity on PC12 cells through co-culturing with RAW264.7 cells

Meth at 100, 200, and 400 µmol/L did not induce toxicity on PC12 cells. Coculturing with RAW264.7 stimulated by LPS/IFNγ slightly decreased the viability of PC12 cells. However, after the addition of Meth in the coculturing system, significant toxicity on PC12 cells was observed (Figure 2(a)), together with the decrease of MMP (Figure (b)), increase in the cytochrome-c releasing into cytoplasm (Figure 2(c)), and the increase in DNA fragmentation in PC12 cells (Figure 2(d)) (p < 0.05).

Figure 2.

methamphetamine (Meth) enhanced the toxicity on PC12 cells after co-culturing with RAW264.7 cells stimulated by IFNγ/LPS. PC12 and RAW264.7 cells were co-cultured using transwell^® 6-well plates and treated with Meth at the concentration of 400 µM at the presence of IFNγ (10 ng/mL)/LPS (100 ng/mL) for 48 h. PC12 cells were collected to test the cytotoxicity. Cytotoxicity were determined by decreasing cell viability (a), decreasing mitochondrial membrane potential (b), increasing cytochrome-c releasing (c), and increasing DNA fragmentation (d). *p < 0.05, **p < 0.01 vs. PC12 cells alone. Samples were measured in triplicate and experiments were repeated three times. LPS: lipopolysaccharide; IFNγ: interferon γ.

Meth increased the IL-6, TNFα, and IL-1β levels and NO production in RAW264.7 cell

Treatment of RAW264.7 cells with LPS/IFNγ increased IL-6, IL-1β, and TNFα levels and NO production in the supernatant (p < 0.01). After combination treatment with Meth, IL-6, IL-1β, and TNFα levels and NO production significantly increased (p < 0.05). However, the addition of D3 receptor antagonist NGB2904 could significantly lower the increase in IL-6, IL-1β, and TNFα levels and NO production caused by Meth treatment (Figure 3).

Figure 3.

Methamphetamine (Meth) promoted polarization of RAW264.7 cells through dopamine receptor D3. RAW264.7 cells were treated with Meth at 400 µmol/L, supplemented with LPS (100 ng/mL) and IFNγ (10 ng/mL), or IL-4 (10 ng/mL) with or without NGB2904 (5 nmol/L) for 48 h. RAW264.7 cells were collected to determine cytokine or molecule levels. Macrophage polarization was determined by different cytokine and molecule releasing: IL-6 (a), TNFα (b), IL-1β (c), NO (d), and IL-10 (e). **p < 0.01 vs. RAW264.7 cells alone; ^# p < 0.05, ^## p < 0.01 vs. RAW264.7 plus IFNγ/LPS or IL-4. Samples were measured in triplicate and experiments were repeated three times. LPS: lipopolysaccharide; IFNγ: interferon γ; TNFα, tumor necrosis factor α; IL, interleukin.

Meth decreases the IL-10 level in RAW264.7 cell

Treatment of RAW264.7 cells with IL-4 increased IL-10 level in the supernatant (p < 0.01). After combination treatment with Meth, IL-10 level significantly decreased (p < 0.05). The addition of D3 receptor antagonist NGB2904 could significantly reverse the decrease in IL-10 level caused by Meth treatment (Figure 3).

Meth increases the mRNA expression of IL-6, TNFα, and IL-1β in RAW264.7 cell

Treatment of RAW264.7 cells with LPS/IFNγ increased mRNA expression of IL-6, TNFα, and IL-1β (p < 0.01). After combination treatment with Meth, IL-6, TNFα, and IL-1β expression dramatically increased (p < 0.01). However, the addition of D3 receptor antagonist NGB2904 could significantly lower the increasing IL-6, TNFα and IL-1β expression caused by Meth treatment (Figure 4).

Figure 4.

Methamphetamine (Meth) promoted polarization of RAW264.7 cells through dopamine receptor D3 by changing gene expression. RAW264.7 cells were treated with Meth at 400 µM, supplemented with LPS (100 ng/mL) and IFNγ (10 ng/mL), or IL-4 (10 ng/mL) with or without NGB2904 (5 nM) for 48 h. RAW264.7 cells were collected to determine gene expression. Macrophage polarization was determined by the gene expression change of different markers: M1 marker (a); M2 marker (b). **p < 0.01 vs. RAW264.7 cells alone; ^## p < 0.01 vs. RAW264.7 plus IFNγ/LPS or IL-4. Samples were measured in triplicate and experiments were repeated three times. LPS: lipopolysaccharide; IFNγ: interferon γ; IL, interleukin

Meth decreases the mRNA expression of Arg-1, IL-10, and KLF4 in RAW264.7 cell

Treatment of RAW264.7 cells with IL-4 increased mRNA expression of Arg-1, IL-10, and KLF4 (p < 0.01). After combination treatment with Meth, expression of Arg-1, IL-10, and KLF4 dramatically decreased (p < 0.01). However, the addition of D3 receptor antagonist NGB2904 could significantly reverse the decrease in Arg-1, IL-10, and KLF4 expression caused by Meth treatment (Figure 4).

Meth changes the protein expression in RAW264.7 cell

Treatment of RAW264.7 cells with LPS/IFNγ increased protein expression of TNFα and IL-1β. Treatment of RAW264.7 cells with IL-4 also increased protein expression of Arg-1 and KLF4. After combination treatment with Meth, TNFα, and IL-1β expression significantly increased, while Arg-1 and KLF4 expression significantly decreased (p < 0.05). The effects caused by Meth could be abolished by the addition of D3 receptor antagonist NGB2904 (Figure 5).

Figure 5.

Methamphetamine (Meth) promoted polarization of RAW264.7 cells through dopamine receptor D3 by changing protein expression. RAW264.7 cells were treated with Meth at 400 µM, supplemented with LPS (100 ng/mL) and IFNγ (10 ng/mL), or IL-4 (10 ng/mL) with or without NGB2904 (5 nM) for 48 h. RAW264.7 cells were collected to determine protein expression. Lane 1: RAW264.7; Lane 2: RAW264.7 + IL-4 (Arg-1 and KLF4) or RAW264.7 + IFNγ/LPS (IL-1β and TNFα); Lane 3: RAW264.7 + IL-4 + Meth (Arg-1 and KLF4) or RAW264.7 + IFNγ/LPS + Meth (IL-1β and TNFα); Lane 4: RAW264.7 + IL-4 + Meth + NGB2904 (Arg-1 and KLF4) or RAW264.7 + IFNγ/LPS + Meth + NGB2904 (IL-1β and TNFα). **p < 0.01 vs. RAW264.7 cells alone; ^# p < 0.05, ^## p < 0.01 vs. RAW264.7 plus IFNγ/LPS or IL-4. Experiments were repeated three times and a representative blot was displayed. LPS: lipopolysaccharide; IFNγ: interferon γ; TNFα, tumor necrosis factor α; IL, interleukin.

Discussion

Meth has been shown to cause neurotoxicity both in vitro and in vivo, and there are no efficacious therapies for this neural injury. Little is known about the chronic Meth-induced neurotoxicity, ^18,19 and this is the first time to explore the mechanism. We demonstrated that Meth at nontoxic concentration caused cell injury through enhancing polarization of macrophages to result in inflammatory responses and apoptosis. This study will provide scientific rationale to the treatment of Meth abuse.

Mitochondria play an important role in the process of cell death regulation. Many genes and proteins can influence or determine the progression of apoptosis along the mitochondrial pathway. ²⁰ The change in MMP could induce the release of cytochrome-c from the mitochondria to nucleus, which activate caspase-related apoptosis protein and facilitate the formation of apoptosome complex, leading to chromatin condensation and DNA cleavage. DNA fragmentation has been considered a biochemical hallmark and widely used as apoptosis index. ^{21 –23} Therefore, the depolarization of MMP and the release of cytochrome-c from mitochondria in PC12 cells were detected to illustrate the onset of apoptosis. ²⁴ In the present study, we found that treatment of PC12 cells with Meth or cocultured RAW264.7 would not decrease cell viability. However, coculturing with RAW264.7 stimulated by IFNγ/LPS and Meth promoted cell injury and DNA fragmentation, accompanied by MMP depolarization and cytochrome-c release in PC12 cells, indicating Meth enhanced cytotoxicity through affecting the macrophages.

Heterogenic macrophages are divided into two subpopulations: classically activated macrophages (M1) and alternatively activated macrophages (M2). ²⁵ LPS and IFNγ activate M1 macrophages, exhibiting high endocytic and phagocytic capacities with high production of IL-12 and toxic intermediates (e.g. NO and reactive oxygen species). M1 macrophages are defined as potent effector cells exhibiting antimicrobial abilities. On the other hand, IL-4, IL-10, or immunoglobulin complexes activate M2 macrophages, which promote tissue repair, and contribute to angiogenesis and tumor progression. ²⁶

Inflammation is a complex reaction of the systemic immune vascularized tissues and accompanied by the activation of various immune cells such as neutrophils, macrophages, and lymphocytes. Particularly, macrophages play important roles in the regulation of inflammation and immune response and are involved in various disease processes. ^{27 –29} Activated macrophages secrete lots of inflammatory mediators such as NO produced by inducible nitric oxide synthase,as well as inflammatory cytokines such as TNFα, IL-1β, and IL-6. ^{30 –32} These cytokines are known as important mediators involved in the progress of many inflammatory diseases. These cytokines and their genes can be modulated by activation of transcription factor nuclear factor κB (NF-κB). Activated NF-κB translocates from cytoplasm to nucleus and binds to promoter and modulates the expression of inflammatory genes. ^33,34

It has been demonstrated that Meth impairs dopaminergic system in both animal model and human drug abusers. ³⁵ Chronic Meth abuse disrupts dopamine homeostasis and damages dopaminergic neurons and terminals. ^36,37 As one of the five dopamine receptors, dopamine receptor D3 is classified as a dopamine 2-like receptor due to its structural and functional similarities. Dopamine receptor D3 is expressed in the cultured primary microglia and serves as a modulator of microglia function during neuroinflammation. Gupta et al. confirmed that dopamine receptor D3 genetic polymorphism was associated with rates of cognitive impairment in methamphetamine-dependent patients with HIV. ^38,39 Exposure to IFNγ and LPS induces M1 polarization which is characterized by the production of NO, TNFα, IL-6, and IL-1β, and relevant genes. All of these contribute to inflammation and exacerbate neuron injury. The present in vitro study confirmed such changes. After Meth treatment, proinflammatory cytokines in the supernatant, including TNFα, IL-6, and IL-1β, and NO increased, while M2 markers and IL-10 decreased, together with the change in gene and protein expression suggesting that Meth could enhance macrophage polarization from M0 to M1, while inhibiting macrophage polarization from M0 to M2. Resultantly, Meth promoted RAW264.7 macrophage polarization and inflammatory response to cause cytotoxicity on PC12 cells in the coculturing system. Moreover, all the effects of Meth on macrophage were antagonized by the dopamine receptor D3 antagonist, indicating Meth exerted its effects through D3 receptor.

In conclusion, our study demonstrated that Meth at subtoxic concentration caused toxicity on neuronal cells through promoting macrophage polarization and inflammatory response. This effect was exerted through the dopamine receptor D3. Our study provided the scientific rationale for the neurotoxicity caused by the chronic use of Meth.

Supplemental material

Supplementary_Material - Methamphetamine causes neurotoxicity by promoting polarization of macrophages and inflammatory response

Supplementary_Material for Methamphetamine causes neurotoxicity by promoting polarization of macrophages and inflammatory response by X Li, F Wu, L Xue, B Wang, J Li, Y Chen and T Chen in Human & Experimental Toxicology