Prevention of methamphetamine-induced microglial cell death by TNF-α and IL-6 through activation of the JAK-STAT pathway

Abstract

Background: It is well known that methamphetamine (METH) is neurotoxic and recent studies have suggested the involvement of neuroinflammatory processes in brain dysfunction induced by misuse of this drug. Indeed, glial cells seem to be activated in response to METH, but its effects on microglial cells are not fully understood. Moreover, it has been shown that cytokines, which are normally released by activated microglia, may have a dual role in response to brain injury. This led us to study the toxic effect of METH on microglial cells by looking to cell death and alterations of tumor necrosis factor-alpha (TNF-α) and interleukine-6 (IL-6) systems, as well as the role played by these cytokines.

Methods: We used the N9 microglial cell line, and cell death and proliferation were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling assay and incorporation of bromodeoxyuridine, respectively. The TNF-α and IL-6 content was quantified by enzyme-linked immunosorbent assay, and changes in TNF receptor 1, IL-6 receptor-alpha, Bax and Bcl-2 protein levels by western blotting. Immunocytochemistry analysis was also performed to evaluate alterations in microglial morphology and in the protein expression of phospho-signal transducer and activator of transcription 3 (pSTAT3).

Results: METH induced microglial cell death in a concentration-dependent manner (EC50 = 1 mM), and also led to significant morphological changes and decreased cell proliferation. Additionally, this drug increased TNF-α extracellular and intracellular levels, as well as its receptor protein levels at 1 h, whereas IL-6 and its receptor levels were increased at 24 h post-exposure. However, the endogenous proinflammatory cytokines did not contribute to METH-induced microglial cell death. On the other hand, exogenous low concentrations of TNF-α or IL-6 had a protective effect. Interestingly, we also verified that the anti-apoptotic role of TNF-α was mediated by activation of IL-6 signaling, specifically the janus kinase (JAK)-STAT3 pathway, which in turn induced down-regulation of the Bax/Bcl-2 ratio.

Conclusions: These findings show that TNF-α and IL-6 have a protective role against METH-induced microglial cell death via the IL-6 receptor, specifically through activation of the JAK-STAT3 pathway, with consequent changes in pro- and anti-apoptotic proteins.

Figure 1

METH induces microglial cell death. (A) METH increased the number of TUNEL-positive cells in a concentration-dependent manner (0.1 to 4 mM for 24 h), and Z-VAD (25 μM) completely prevented the apoptotic cell death induced by 1 mM METH. The results are expressed as percentage of total cells ± SEM (n = 10 to 25). ^***P <0.001, Dunnett’s multiple comparison test, significantly different when compared to control. ⁺⁺⁺P <0.001 to Bonferroni’s multiple comparison test, significantly different comparing with 1 mM METH. (B) Representative fluorescence images of TUNEL-positive cells following treatment with 0.1, 1 or 4 mM METH. Scale bar, 20 μm. METH: methamphetamine; SEM: standard error of the mean; TUNEL: terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay; Z-VAD: z-Val-Ala-DL-Asp (OMe)-fluoromethylketone.

Figure 2

METH causes microglial activation and cytoskeleton re-organization. Representative confocal images of F-actin (red) and Iba-1 immunoreactivity (green) in N9 microglial cells under control conditions and exposed to 1 mM METH. Cells were also stained with Hoechst 33342 (blue). Scale bar, 20 μm and 50 μm. Iba-1: ionized calcium binding adaptor molecule-1; METH: methamphetamine.

Figure 3

METH affects microglial proliferation. METH increased the number of BrdU-positive cells at 0.001 and 0.01 mM, but had a negative effect at 0.1 and 1 mM. Z-VAD (25 μM) reduced, but did not completely prevent, the decrease in the number of BrdU-positive cells induced by METH. The results are expressed as percentage of total cells ± SEM (n = 10 to 35). ^***P <0.01, ^***P <0.001, Dunnett’s multiple comparison test, significantly different comparing to control. ⁺⁺⁺P <0.001, Bonferroni’s multiple comparison test, significantly different comparing with 1 mM METH. BrdU: 5-bromo-2’-deoxyuridine; METH: methamphetamine; SEM: standard error of the mean; Z-VAD: z-Val-Ala-DL-Asp (OMe)-fluoromethylketone.

Figure 4

METH triggers an early increase in TNF-α protein levels. The effect of 1 mM METH on the (A, C) extracellular and (B, D) intracellular TNF-α levels was evaluated after (A, B) 1 h and (C, D) 24 h of drug exposure by ELISA. As a positive control we used 1 μg/mL LPS. Data are expressed as mean ± SEM of pg/mL for release and pg/mg of total protein for the intracellular levels (n = 3 to 13). ^**P <0.01, ^***P <0.001, Dunnett’s post-test, significantly different from control. ELISA: enzyme-linked immunosorbent assay; LPS: lipopolysaccharide; METH: methamphetamine; SEM: standard error of the mean; TNF-α: tumor necrosis factor-alpha.

Figure 5

METH increases IL-6 protein levels after 24 hours. METH did not interfere with the (A) extracellular and (B) intracellular levels of IL-6 when analyzed 1 h post-treatment. However, following 24 h of METH exposure there was a significant increase in the (C) extracellular and (D) intracellular levels of IL-6. The treatment with (C, D) 1 μg/mL LPS was used as a positive control and up-regulated IL-6 levels after 24 h. Data are expressed as mean ± SEM of pg/mL for extracellular levels and pg/mg of total protein for intracellular levels (n = 4-15). ^*P <0.05, ^**P <0.01, ^***P <0.001, Dunnett’s post-test, significantly different from control. IL-6: interleukin-6; LPS: lipopolysaccharide; METH: methamphetamine; SEM: standard error of the mean.

Figure 6

METH increases TNFR1 and IL-6R-α protein levels. Quantification of (A) TNFR1 and (B) IL-6R-α protein levels in N9 microglial cells at 1 h and 24 h post-METH exposure, respectively. Above the bars, representative western blot images of TNFR1 (55 kDa), IL-6R-α (63 kDa) and actin (42 kDa) are shown. The results are expressed as mean percentage of control ± SEM (n = 3 to 15). ^*P <0.05, Mann–Whitney post-test, significantly different from control. IL-6R-α: interleukin-6 receptor-alpha; kDa: kiloDaltons; LPS: lipopolysaccharide; METH: methamphetamine; SEM: standard error of the mean; TNFR1: tumor necrosis factor receptor 1.

Figure 7

Endogenous TNF-α and IL-6 are not involved in METH-induced microglial cell death. The increase in the number of TUNEL-positive cells induced by 1 mM METH (24 h) was not changed by (A) TNF-α antibody or (B) IL-6R antibody, and the JAK-STAT pathway inhibitor (20 μ AG 490) exacerbated the toxic effect of METH. The results are expressed as mean percentage of total cells ± SEM (n = 17 to 84). ^***P <0.001, Dunnett’s post-test, significantly different when compared with control. ⁺⁺P <0.01, Bonferroni’s post-test, when compared with 1 mM METH. IL-6: interleukin 6; IL-6R: interleukin 6 receptor; JAK-STAT: janus kinase-signal transducer and activator of transcription; METH: methamphetamine; SEM: standard error of the mean; TNF-α: tumor necrosis factor-alpha; TNFR1: tumor necrosis factor receptor 1.

Figure 8

Protective effect of exogenous TNF-α or IL-6 against METH-induced microglial cell death. (A) TNF-α (1 ng/mL) completely prevented the increase of TUNEL-positive cells induced by METH; this was abolished by IL-6R antibody (10 μg/mL). (B) IL-6 (1 ng/mL) reduced the number of TUNEL-positive cells, and once again the blockade of its receptor with IL-6R antibody abolished this effect. Also, the JAK-STAT3 inhibitor (20 μM AG 490) exacerbated METH toxicity. The results are expressed as mean percentage of total cells ± SEM (n = 17 to 41). ^***P <0.001, Dunnett’s multiple comparison test, significantly different when compared to control; ⁺⁺P <0.01, ⁺⁺⁺P <0.001, Bonferroni’s multiple comparison test, significantly different when compared with 1 mM METH; ^§§§P <0.001, Bonferroni’s multiple comparison test, significantly different when compared with 1 mM METH plus TNF-α or IL-6. (C) Representative images of pSTAT3 immunoreactivity (red) in N9 microglial cells under control conditions and exposed to 1 mM METH alone or in the presence of 1 ng/mL IL-6. Cells were also stained with Hoechst 33342 (blue). Scale bar, 20 μm and 50 μm. IL-6: interleukin 6; JAK-STAT: janus kinase-signal transducer and activator of transcription; METH: methamphetamine; pSTAT: phospho-signal transducer and activator of transcription; SEM: standard error of the mean; TNF-α: tumor necrosis factor-alpha; TUNEL: terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay.

Figure 9

IL-6 prevents the increase of the Bax/Bcl-2 ratio mediated by METH. Western blot analysis shows that IL-6 (1 ng/mL) was able to reduce the augmentation of the Bax/Bcl-2 ratio following METH exposure. Above the bars, representative western blot images of Bax (21 kDa), Bcl-2 (26 kDa) and β-actin (42 kDa) are shown. The results are expressed as mean percentage of control ± SEM (n = 4 to 7). ^*P <0.05, Dunnett’s multiple comparison test, significantly different when compared with control; ⁺P <0.05, Bonferroni’s multiple comparison test, significantly different when compared with 1 mM METH. IL-6: interleukin 6; kDa: kiloDaltons; METH: methamphetamine; SEM: standard error of the mean.