Minocycline suppresses hypoxic activation of rodent microglia in culture

Neuroscience Letters 366 (2004) 167–171

Minocycline suppresses hypoxic activation of rodent microglia in culture夽 Kyoungho Suk∗ Department of Pharmacology, School of Medicine, Kyungpook National University, #101 Dong-In, Joong-gu, Daegu 700-422, South Korea Received 13 April 2004; received in revised form 8 May 2004; accepted 12 May 2004

Abstract Hypoxia is one of the important physiological stimuli that are often associated with a variety of pathological states such as ischemia, respiratory diseases, and tumorigenesis. In the central nervous system, hypoxia that is accompanied by cerebral ischemia not only causes neuronal cell injury, but may also induce pathological microglial activation. We have previously shown that hypoxia induces inflammatory activation of cultured microglia, and the hypoxic induction of nitric oxide production in microglia is mediated through p38 mitogen-activated protein kinase pathway. Now, we present evidence that minocycline, a tetracycline derivative, suppresses the hypoxic activation of cultured microglia by inhibiting p38 mitogen-activated protein kinase pathway. The drug markedly inhibited hypoxia-induced production of inflammatory mediators such as nitric oxide, TNF␣, and IL-1␤ as well as iNOS protein expression. The signal transduction pathway that leads to the activation of p38 mitogen-activated protein kinase was the molecular target of minocycline. Thus, the known neuroprotective effects of minocycline in animal models of cerebral ischemia may be partly due to its direct actions on brain microglia. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Minocycline; Hypoxia; Microglia; Inflammation; Ischemia; Mitogen-activated protein kinase

Minocycline is a semi-synthetic second-generation tetracycline that exerts anti-inflammatory effects unrelated to its anti-microbial action [2,15]. The drug is currently considered for treatment of inflammatory diseases such as rheumatoid arthritis. It has been previously reported that minocycline attenuates the death of hippocampal neurons in a gerbil model of global ischemia [25], and it is neuroprotective against focal ischemia in rats [26]. In these animal models of ischemia, minocycline inhibited microglial activation and the production of inflammatory mediators. Minocycline also provided neuroprotection against excitotoxicity by inhibiting activation and proliferation of microglia [20,21]. Activated microglia are believed to contribute to neuronal damage in stroke through the excess release of proinflammatory and/or cytotoxic factors [5]. Thus, inhibition of microglial activation may provide a means of therapeutic intervention against ischemic stroke, where inflammatory activation of microglia plays a 夽 Supplementary data associated with this article can be found at doi:10.1016/j.neulet.2004.05.038. ∗ Tel.: +82 53 420 4835; fax: +82 53 256 1566. E-mail address: [email protected] (K. Suk).

pathogenic role. Neurons and kidney epithelial cells were also target of minocycline action. Treatment with minocycline salvaged cultured primary neurons from glutamate- or 6-hydoxydopamine-induced toxicity [12,26]. Minocycline protected kidney epithelial cells in vitro and protected the kidneys from ischemic injury in vivo [22]. Previously, we have demonstrated that hypoxia induces inflammatory activation of cultured microglia, and the hypoxic induction of nitric oxide production in microglia is mediated through p38 MAPK pathway [14]. During cerebral ischemia, hypoxia may not only impose the damages on neurons directly, but also promote neuronal injury indirectly via microglial activation. In the current studies, we examined whether neuroprotective minocycline acts on microglia to inhibit their activation under hypoxic condition. Isolated microglia cultures were used to assess the effects of minocycline on microglia in the absence of neurons or other types of glial cells. Although neuroprotective effects of minocycline have been well demonstrated in several neural injury models in vivo [3,11,12,16,18,19], its effects on cultured microglia that were activated by hypoxic stimuli have not been studied. Moreover, in animal models, the primary effect of the drug on microglia can not be accurately

0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.05.038

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determined because of the co-existence of other cell types. Using rat primary cultures of microglia and a pure mouse microglial cell line, we have demonstrated that minocycline inhibits hypoxic activation of microglia and the p38 MAPK pathway appears to be one of the targets of minocycline action in microglia. Minocycline was obtained from Sigma (St. Louis, MO). BV-2 mouse microglia cell line originally developed by Dr. V. Bocchini at University of Perugia (Perugia, Italy) [4] was maintained as previously described [9]. Rat primary microglial cultures were prepared as previously described with minor modifications [1,10]. In brief, forebrains of newborn Sprague–Dawley rats were chopped and dissociated by trypsinization and mechanical disruption. The cells were seeded into poly-l-lysine-coated flasks. After in vitro culture for 10 days, microglial cells were detached by rapid and gentle shaking of the culture flasks and seeded into plastic surfaces. After additional 1 h incubation, non-adherent cells were removed by replacing culture medium. The purity of microglial cultures was greater than 90% as determined by OX-42 immunocytochemical staining (data not shown). The experimental protocols for the laboratory animals were in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised in 1996. Adequate measures were taken to minimize pain or discomfort of animals. For the generation of hypoxic condition, cells were washed three times with deoxygenated serum-free DMEM, and then immediately placed into an anaerobic chamber (O2 tension < 0.2%) (Anaerobic System 1025, Forma Scientific Inc., Marietta, OH) and incubated at 37 ◦ C within the chamber. To reoxygenate hypoxic cultures, cells were transferred into a regular normoxic incubator (95% air, 5% CO2 ) and incubation continued for the indicated time periods. Normoxic control cells were washed with a normal serum-free DMEM and incubated under normoxic condition for the corresponding time periods. For Western blot analysis, only hypoxia was delivered for 8 h (for iNOS) or 1 h (for p38 MAPK) without reoxygenation. Cytotoxicity was assessed by MTT assays as described [17]. In brief, cells were seeded in 96-well plates and exposed to hypoxia-reoxygenation with or without minocycline. After the treatment, the medium was removed and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, 0.5 mg/ml) was added, followed by incubation at 37 ◦ C for 2 h in CO2 incubator. After a brief centrifugation, supernatants were carefully removed and DMSO was added to the cells. After insoluble crystals were completely dissolved, absorbance at 540 nm was measured using Thermomax microplate reader (Molecular Devices). NO2 − in culture supernatants was measured to assess NO production in microglial cells as previously described [8]. NaNO2 was used as the standard to calculate NO2 − concentrations. TNF␣ and IL-1␤ secreted in microglial culture supernatants was measured by specific ELISA as previously described [8].

For Western blot analysis, cells were lysed in tripledetergent lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.02% sodium azide, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 1 mM PMSF). Protein concentration in cell lysates was determined using Bio-Rad protein assay kit. An equal amount of protein for each sample was separated by 8% or 12% SDS–PAGE and transferred to Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK). The membrane was blocked with 5% skim milk and incubated with rabbit polyclonal antibodies specific for either iNOS (Transduction Laboratories, San Diego, CA), phosphorylated (Thr180/Tyr182) or total p38 MAPK (Cell Signaling, Beverly, MA). Then, incubation with horseradish peroxidase-conjugated anti-rabbit antibodies and ECL detection (Amersham Pharmacia Biotech) followed. Statistical comparison between different treatments was done by either Student’s t-test or one-way ANOVA with Dunnet’s multiple comparison test using GraphPad Prism program (GraphPad Software Inc., San Diego, CA). Differences with P value less than 0.05 were considered statistically significant. To determine whether minocycline regulates hypoxic activation of microglia, primary microglia cultures prepared from neonatal rat brain were exposed to hypoxia/reoxygenation in the presence or absence of minocycline, and the amount of NO produced was measured as an indicator of microglial activation. Compared to normoxic cells, hypoxia/reoxygenation strongly induced NO production in microglia cultures (6.3-fold increase) (Fig. 1A). Reoxygenation for 24 h was required for NO production [14]. Under hypoxic condition (without reoxygenation), production of NO was not achieved possibly due to insufficient oxygen tension and consequent impairment of the oxidation of l-arginine [13]. Hypoxia-induced NO production was markedly reduced by minocycline pretreatment (1–10 ␮M). Minocycline at 1 or 10 ␮M did not exert a significant cytotoxicity as assessed by MTT assays (101.2 ± 0.6% viability at 1 ␮M or 98.5 ± 0.7% viability at 10 ␮M, when the viability of untreated control cells was set to 100%). Hypoxic induction of NO production and its inhibition by minocycline were similarly observed in a pure microglial cell line (Fig. 1B), indicating that the observed effects of minocycline on primary microglia cultures are solely based on microglia, but not contaminating astrocytes in the primary cultures. Hypoxic induction of NO production was mainly due to iNOS induction, which was also diminished by minocycline (Fig. 1A, inset). Hypoxic activation of microglia and the inhibition by minocycline were confirmed on the basis of morphological changes of microglia (Fig. 1C). When the production of TNF␣ or IL-1␤ was next assessed, hypoxia was also found to trigger production of these inflammatory cytokines in microglia, and a similar inhibitory effect of minocycline was observed (Fig. 2). Thus, hypoxic condition could lead to inflammatory activation of isolated microglia, and minocycline suppresses this. In addition, the suppression of inflammatory

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Fig. 1. Hypoxic induction of NO and iNOS in microglia and their inhibition by minocycline. Microglia cultures isolated from rat brain (A) or BV-2 mouse microglial cells (B) were exposed to hypoxia for 8 h and reoxygenated for 24 h in the presence or absence of minocycline (0.1–10 ␮M), and then NO production was evaluated by Griess reaction. Minocycline (0.1–10 ␮M) under normoxic condition did not induce a significant NO production (data not shown). Normoxia represents average NO production by microglia cultured under normoxic condition for the corresponding time periods, which was set to 1. Microglia were pretreated with minocycline for 1 h before the exposure to hypoxia. The results are mean ± S.D. (n = 3). Asterisks indicate statistically significant differences from hypoxic condition without minocycline (P < 0.05). The level of iNOS protein was measured by Western blot in primary microglia cultured under a similar hypoxic condition (8 h) (A inset). The equal loading of samples was confirmed by Ponceau-S staining of total proteins (data not shown). N, normoxia; H, hypoxia; M + H, hypoxia in the presence of minocycline (10 ␮M). The effects of minocycline on microglial activation based on morphological changes are also shown (C) (original magnification, 400×). Hypoxia-induced deramification of microglia, which was partly reversed by minocycline (10 ␮M).

activation of microglia by minocycline protected microglia from inflammatory injury. Exposure of microglia to hypoxia (8 h) followed by reoxygenation (24 h) caused a significant reduction in their viability (27.9 ± 1.9% viability compared to untreated control that was set to 100%) [14]; and this was partly reversed by minocycline (10 ␮M) pretreatment (52.3 ± 2.1% viability). The reduction in the cell viability appears to be due to large amounts of NO or other inflammatory mediators produced, which are known to cause the apoptosis of microglia in an autocrine manner [9,10].

Fig. 2. Inhibition by minocycline of hypoxic induction of TNF␣ or IL-1␤ production in microglia. After BV-2 cells were exposed to hypoxia for 8 h and reoxygenated for 24 h with or without minocycline pretreatment (1 h, 10 ␮M), TNF␣ or IL-1␤ production was assessed by ELISA. The results are mean ± S.D. (n = 3). Asterisks indicate statistically significant differences between two treatments (P < 0.05). Nor, normoxia; Hyp, hypoxia; Mino + Hyp, hypoxia with minocycline pretreatment.

As we have previously shown that hypoxic activation of microglia is mediated through p38 MAPK [14], we next asked whether treatment with minocycline influences the hypoxic activation of the kinase. While the activation of p38 MAPK is an early step in the microglial signal transduction, the expression of iNOS protein is a downstream effect. In our previous report [14], we have conducted a time course study on p38 MAPK activation after hypoxia. The activation of p38 MAPK peaked at 60 min after hypoxia and decreased thereafter. Therefore, we selected a time point of the peak activation or induction of microglial signaling molecules to assess the effects of minocycline. Hypoxia for 1 h strongly induced activation of p38 MAPK in rat microglia cultures as demonstrated by the appearance of phosphorylated form of the kinase (Fig. 3). Reoxygenation, however, did not induce the kinase activation (data not shown), suggesting that hypoxia alone is sufficient to induce microglial activation. The hypoxic activation of p38 MAPK was significantly inhibited by minocycline pretreatment (78.6% inhibition based on the densitometric analysis of Western blot results), indicating that p38 MAPK pathway may be one of targets of minocycline action in microglia. However, it remains to be determined which step(s) in the signal transduction pathway that leads to p38 MAPK activation is inhibited by minocycline in microglia. In this work, we present evidence that inflammatory activation of microglia under hypoxic condition can be inhibited by minocycline. The inhibitory effect of minocy-

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References

Fig. 3. Inhibition of hypoxic activation of p38 MAPK by minocycline. After pretreatment with or without minocycline (10 ␮M) for 1 h, primary microglia cells were cultured under hypoxic condition for 1 h, then phosphorylated p38 MAPK (p-p38) or total p38 MAPK (p38) was detected by Western blot analysis using specific antibodies. The results are representative of three independent experiments.

cline was accompanied by suppression of p38 MAPK activation. Neuroprotective effects of minocycline have been previously reported in animal models of ischemia [25,26] and neurodegenerative diseases [7,19,23,24,27]. The role of minocycline as a neuroprotector was associated with inhibition of microglial activation in some of these previous studies [7,24–26]. However, the effect of minocycline on microglia cultured under hypoxic condition has not been assessed. Now, our findings that minocycline suppresses hypoxic activation of cultured microglia suggest that minocycline may directly act on microglia to exert its neuroprotective effects against hypoxia/ ischemia. The fact that microglia can be directly activated by hypoxia suggests an important role of microglia in neuronal injury during cerebral ischemia. Although it has been previously thought that neuroglial cells are activated by signals originated from dying neurons under pathological condition in CNS [6], our current findings that hypoxia could activate isolated microglia cultures as well as a microglial cell line to produce inflammatory mediators suggest that hypoxia may initiate inflammation by direct activation of microglia independently of neuronal cell death. Hypoxia seems to influence both neurons and microglia leading to pathological death and inflammatory activation, respectively. Nevertheless, based on our current findings, the primary target of minocycline action against hypoxia/ischemia seems to be microglia.

Acknowledgements This work was supported by grants from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (03-PJ1-PG10-21300-0011).

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