Original Paper Received: April 17, 2014 Accepted after revision: June 19, 2014 Published online: October 3, 2014
Neuroimmunomodulation DOI: 10.1159/000365484
Lipopolysaccharide-Stimulated Transglutaminase 2 Expression Enhances Endocytosis Activity in the Mouse Microglial Cell Line BV-2 Kenji Kawabe Katsura Takano Mitsuaki Moriyama Yoichi Nakamura Laboratory of Integrative Physiology in Veterinary Sciences, Osaka Prefecture University, Izumisano, Japan
Abstract Objectives: In peripheral macrophages, tissue-type transglutaminase (TG2) is reported to be involved in phagocytosis of apoptotic cells. However, the contribution of TG2 to microglial phagocytosis has not been investigated. In this study, using a microglial cell line, BV-2, we examined the changes in TG2 expression, phagocytosis and pinocytosis in cells stimulated by lipopolysaccharide (LPS). Methods: Cells of the mouse microglial cell line BV-2 were stimulated by LPS with or without cystamine, an inhibitor of TG enzyme activity, for 24 h. TG2 expression was measured by real-time RTPCR and Western blotting. TG activity was evaluated using biotinylated pentylamine as a substrate. Pinocytosis was determined by uptake of 1-μm fluorescent microbeads. Phagocytosis was assessed by uptake of dead cells, human neuroblastoma SH-SY5Y cells, which were pretreated with H2O2 for 24 h. Results: Phagocytosis of dead cells and pinocytosis of fluorescent microbeads were up-regulated by LPS stimulation together with TG2 expression. Blockade of TG enzyme activity by cystamine suppressed TG2 expression, phagocytosis and pinocytosis. Conclusions: These results suggested that LPS-induced TG2 was involved in the mechanism of pinocytosis and phagocytosis in microglia. © 2014 S. Karger AG, Basel
© 2014 S. Karger AG, Basel 1021–7401/14/0000–0000$39.50/0 E-Mail [email protected] www.karger.com/nim
Introduction
Microglia categorized into a type of macrophages are considered to be a major cell for immunity in CNS. Activated microglia release nitric oxide (NO) and proinflammatory cytokines to damage neurons, and also produce neurotrophins to protect neurons. In addition, they engulf invading microorganisms and scavenge cell debris and damaged cells [1, 2]. Microglial phagocytosis contributes to the maintenance of CNS homeostasis and the development of a neural network physiologically. However, it is reported that NO production and phagocytosis of hyperactivated microglia cause neuronal death in neurodegenerative diseases [3]. Several studies found that tissue-type transglutaminase (TG2) is overexpressed in brains affected with Alzheimer’s, Parkinson’s and Huntington’s diseases [4, 5], indicating that TG2 plays an important role in CNS diseases. However, there are few reports on microglial TG2 functions. TG2 is ubiquitously expressed in various cells and shows Ca2+ concentration-dependent enzyme activity; it catalyzes protein cross-linking between glutamine and lysine residues of proteins to form ε-(γ-glutamyl)lysine isopeptide bonds [4, 5]. In addition, TG2 protein has binding domains to integrin and fibronectin in a Ca2+-independent manner [4, 5]. These functions contribute to extracellular matrix formation, tissue structure stabilization and epithelia barrier functions. Moreover, TG2 is reported to act also as a G-protein, protein disulfide isomDr. Katsura Takano Laboratory of Integrative Physiology in Veterinary Sciences, Osaka Prefecture University 1-58, Rinku-Ourai Kita Izumisano, Osaka 598-8531 (Japan) E-Mail takano @ vet.osakafu-u.ac.jp
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Key Words Cystamine · Endocytosis · Microglia · Phagocytosis · Pinocytosis · Transglutaminase 2
erase and protein kinase, mediating various intracellular signaling pathways [4–7]. These various functions of TG2 contribute to proliferation, apoptosis, migration and phagocytosis, for example [4, 5]. When phagocytes engulf dead/apoptotic cells, they need to interact with the ‘eat-me’ signal, such as phosphatidylserine (PS), which is exposed on the surface of target cells [8, 9]. It is suggested that TG2 protein in peritoneal macrophages is involved in the recognition of apoptotic cells as a result of the reduced phagocytic capacity in TG2 null macrophages [10, 11]. TG2 might be associated with microglial phagocytosis as well as peritoneal macrophages. In the present study, we examined the effect of lipopolysaccharide (LPS) stimulation on phagocytotic activity of microglia, as well as on TG2 expression and TG enzyme activity in the mouse microglial cell line BV-2.
Experimental Procedures Cell Culture Mouse microglial cell line BV-2 cells were grown and maintained in Dulbecco’s modified Eagle medium (DMEM; Gibco, Grand Island, N.Y., USA) supplemented with 10% fetal bovine serum (Nichirei, Tokyo, Japan) and penicillin/streptomycin at 37 ° C in a humidified incubator under 5% CO2. Cells were subcultured once a week. BV-2 cells were kindly provided by the Laboratory of Molecular Pharmacology, Kanazawa University Graduate School.
Real-Time RT-PCR For real-time RT-PCR, BV-2 cells (2 × 105 cells/well) were replated onto non-coating 24-well plates (Sumilon; Sumitomo Bakelite Co., Tokyo, Japan) and stimulated with LPS (from Salmonella serovar Enteritidis or Escherichia coli; Sigma, St. Louis, Mo., USA) for 24 h. Total RNA was isolated using a FavorPrepTM tissue total RNA purification mini kit (Favorgen, Ping-Tung, Taiwan, ROC). Complementary DNA was prepared using an Omniscript reverse transcription kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. PCR experiments were done using a SYBR Green real-time PCR master mix (Toyobo, Osaka, Japan) for TG2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primer sequences used were: TG2 sense strand (5′-ACT TCG ACG TGT TTG CCC ACA T-3′), TG2 antisense strand (5′-TTG ATG TCC TCA GTG CCA CAC T-3′); GAPDH sense strand (5′TTG TCA GCA ATG CAT CCT GC-3′), and GAPDH antisense strand (5′-AAT GGG AGT TGC TGT TGA AGT C-3′; Operon, Tokyo, Japan). The PCR products are 127 bp for TG2 and 194 bp for GAPDH. The conditions of each PCR cycle for these primers were as follows: denaturation at 95 ° C for 15 s; annealing at 65 ° C for 15 s, and extension at 60 ° C for 30 s. Detection of the fluorescent product was carried out at the end of the extension period. To compare mRNA levels among samples, mRNA of each gene of interest was normalized to the expression of a housekeeping gene, GAPDH, using the comparative Ct method. The changes in the levels of each PCR product were calculated as percent of control.
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Neuroimmunomodulation DOI: 10.1159/000365484
TG Activity Assay Total TG activity was determined using a method developed by Beck et al. [12] with modifications. BV-2 cells (8 × 105 cells/dish) were plated onto non-coating 35-mm dishes and stimulated with 300 ng/ml LPS for 24 h in the presence of 250 μM biotinylated pentylamine (Thermo Fisher Scientific, Waltham, Mass., USA) as the substrate for TG enzyme. Cells were washed, harvested and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by membrane blotting similarly to the Western blotting assay described above. After blocking treatment, the membrane was reacted with 2.5 μg/ml NeutrAvidin-HRP (Thermo Fisher Scientific) dissolved in blocking buffer for 1 h, and TG activity was detected with HRP substrate. Protein profiles were analyzed using a lumino image analyzer (LAS-4000). The total density of 25- to 250-kDa detection bands in each lane measured by ImageJ software was evaluated. Nitrite Assay NO released from BV-2 cells was analyzed by assaying the levels of nitrite (NO2–), a relatively stable metabolite of NO. Amounts of NO2– accumulated in the medium were determined by a fluorometric method using 2,3-diaminonaphthalene (DAN) (Dojindo, Kumamoto, Japan), as described previously [13]. BV-2 cells (4 × 104 cells/well) replated onto 96-well plates were stimulated with 300 ng/ml LPS for 24 h. A 100-μl aliquot of cell-free supernatant was mixed with 20 μl of DAN solution; DAN stock solution (5 mg/ ml in 0.62 M HCl) was freshly diluted 100 times with 0.62 M HCl. The reaction was stopped with 100 μl of 0.28 M NaOH after a 10min incubation at room temperature, and the intensity of the fluorescent product, 2,3-diaminonaphthotriazole, was measured using
Kawabe/Takano/Moriyama/Nakamura
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Western Blotting For Western blotting, BV-2 cells (8 × 105 cells/dish) were replated onto non-coating 35-mm dishes (Sumilon) and stimulated with LPS for 24 h. Cells were homogenized in 20 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA and protease inhibitor cocktail (Sigma P8340). Each homogenate was mixed with one fourth volume of 50 mM Tris-HCl buffer (pH 6.8) containing 50% glycerol, 10% sodium dodecyl sulfate, 0.05% bromophenol blue and 25% 2-mercaptoethanol, followed by boiling at 100 ° C for 5 min. Each aliquot in a certain amount of protein was loaded onto a 10% polyacrylamide gel for electrophoresis at a constant voltage of 120 V for 2.5 h and subsequent blotting to a polyvinylidene fluoride membrane previously treated with 100% methanol. After blocking with 5% skimmed milk dissolved in 20 mM Tris-HCl buffer (pH 7.5) containing 137 mM NaCl and 0.05% Tween 20, the membrane was reacted with antibodies against TG2 (1: 500; Abcam, Cambridge, UK), induced NO synthase (iNOS; 1:1,000, from Dr. Ihara, Osaka Prefecture University, Graduate School of Science) or β-actin (1: 100,000; Sigma) followed by a reaction with anti-mouse IgG antibody (1: 10,000; Bio-Rad, Hercules, Calif., USA) conjugated with horseradish peroxidase (HRP). Proteins reactive with those antibodies were detected using ImmobilonTM Western chemiluminescent HRP substrate (Millipore, Billerica, Mass., USA). Detection bands were assessed using a lumino image analyzer (LAS-4000; Fujifilm, Tokyo, Japan). The ratios of TG2/βactin or iNOS/β-actin of detection bands were evaluated. Protein concentrations were determined by the method using CBB color solution (Nacalai Tesque, Kyoto, Japan), according to the manufacturer’s protocol, with bovine serum albumin as standard.
Data Analysis For statistical analysis of the data, one-way ANOVA followed by Tukey’s multiple comparison procedure or Student’s t test was used. Differences between treatments were considered statistically significant when p < 0.05.
Results
Up-Regulation of TG2 Expression by LPS in BV-2 Cells We examined the effect of LPS on TG2 expression in BV-2 cells. When the cells were stimulated with various concentrations of LPS for 24 h, TG2 mRNA expression increased in a concentration-dependent manner; significant increases were observed with more than 100 ng/ml LPS (fig. 1a). Protein expression of TG2 was also augmented markedly by 100 and 300 ng/ml LPS (fig. 1b). Blockade of LPS-Induced TG2 Expression and TG Activity by a TG Inhibitor We assessed the effect of cystamine, an inhibitor of TG enzyme activity, on TG2 expression. The cells were stimulated by 300 ng/ml LPS with or without 1 mM cystamine for 24 h, and then we assessed the expression levels of TG2 mRNA and protein. Cystamine significantly suppressed LPS-Stimulated TG2 Expression Enhances Phagocytosis in Microglia
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tion. BV-2 cells were stimulated by various concentrations of LPS for 24 h. a mRNA expression of TG2 and GAPDH was assessed by real-time RT-PCR. b TG2 protein expression was detected by Western blotting. Typical bands of Western blotting for TG2 and β-actin proteins were shown in the photograph. The graph shows the TG2/β-actin ratio of the density of detection bands. Data are means ± SD of 3 samples. * p < 0.05, ** p < 0.01, vs. LPS 0 ng/ml.
LPS-induced TG2 mRNA (fig. 2a) and protein (fig. 2b) expression. In order to confirm that cystamine actually inhibited TG enzyme activity under the conditions used, we assessed total TG activity during LPS stimulation for 24 h. TG enzyme activity increased significantly following stimulation with 300 ng/ml LPS. In the presence of 1 mM cystamine, the LPS-induced elevation in TG enzyme activity was depressed (fig. 3). Effects of Cystamine on LPS-Induced iNOS Expression and NO Production We examined whether LPS-induced TG expression and activity were related to the induction of iNOS and Neuroimmunomodulation DOI: 10.1159/000365484
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Endocytosis Assay BV-2 cells (2 × 105 cells/well) were replated onto non-coating 24-well plates and stimulated with LPS for 24 h. Pinocytosis was assessed by uptake of small-sized microbeads (diameter = 1 μm; FluoSphere®; carboxylate modified fluorescent microspheres, orange fluorescent; Molecular Probes, Eugene, Oreg., USA). After stimulation, microbeads (0.013% solid) were added to BV-2 cells and incubated for 1 h. Then BV-2 cells were washed with DMEM 3 times and fixed with 4% paraformaldehyde for fluorescent microscopy. After taking photographs, more than 20 pinocytotic cells were picked up to measure cellular fluorescence intensity by ImageJ software, and average intensity was evaluated. Phagocytosis was assessed by uptake of dead cells. Human neuroblastoma SH-SY5Y cells were treated with 200 μM H2O2 for 24 h to induce apoptosis. We confirmed that the treated cells were apoptotic exposing PS on cell surface with fluorescein isothiocyanate conjugated annexin V (Medical and Biological Laboratories, Nagoya, Japan). Apoptotic cells were stored at –80 ° C. Before use, dead cells were thawed and stained with 10 μg/ml propidium iodide for 30 min at room temperature; then the stained dead cells (2 × 106 cells/well) were added to BV-2 cells and incubated for 30 min. Then BV-2 cells were washed with DMEM three times and fixed with 4% paraformaldehyde. After taking photographs of phase-contrast and fluorescence images, total BV-2 cells and fluorescence superimposed BV-2 cells were counted.
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expression by cystamine. BV-2 cells were stimulated by 300 ng/ml LPS for 24 h in the presence or absence of 1 mM cystamine. a mRNA expression of TG2 and GAPDH was assessed by real-time RT-PCR. b TG2 protein expression was detected by Western blotting. The results are shown in the photograph and graph similar to figure 1b. Data are means ± SD of 3–4 samples. * p < 0.05, ** p < 0.01, vs. LPS 0 ng/ml, # p < 0.05, ## p < 0.01, vs. LPS 300 ng/ml.
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activities of NO production. Protein expression of iNOS was remarkably up-regulated by 300 ng/ml LPS, and 1 mM cystamine blocked LPS-induced up-regulation of iNOS expression to the basal level (fig. 4a). LPS-evoked NO production was also completely suppressed by 1 mM cystamine (fig. 4b). Effects of Cystamine on LPS-Enhanced Endocytosis We assessed two types of endocytosis activities with fluorescent small-sized microbeads (1 μm in diameter) and apoptotic/dead cells: pinocytosis and phagocytosis, respectively (for details, see Discussion). The percentages of pinocytosis action cells were about 30∼50% of total cells and not significantly different with LPS and cysta4
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tivity by cystamine. TG activity was assayed using biotinylated pentylamine (see Experimental Procedures). The photograph shows a typical protein profile produced by TG enzyme activity. The graph shows the total density of 25- to 250-kDa detection bands in each lane. Data are means ± SD of 3 samples. ** p < 0.01, vs. LPS 0 ng/ml, # p < 0.05, vs. LPS 300 ng/ml.
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mine stimulation, but pinocytosis activities of each cell changed remarkably. Fluorescence of microbeads in LPSstimulated cells increased in a concentration-dependent manner, with significant differences at concentrations of 30–300 ng/ml compared with control cells without LPS stimulation. The LPS-enhanced fluorescence was suppressed in the presence of 1 mM cystamine, and levels returned almost to the control level (fig. 5a). In control BV-2 cells, a small percentage of cells showed phagocytotic action, incorporating dead cells. However, when the cells were stimulated with 300 ng/ml LPS, more than 20% of the cells were able to incorporate dead cells. Some BV-2 cells took up more than 2 dead cells. The LPS-enhanced dead cell incorporation was Kawabe/Takano/Moriyama/Nakamura
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Fig. 2. Inhibition of LPS-stimulated TG2
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Fig. 5. Inhibition of LPS-stimulated endocytosis by cystamine. BV-2 cells were stimulated by various concentrations of LPS for 24 h in the presence or absence of 1 mM cystamine before the following uptake measurements. a Uptake of fluorescent microbeads for 1 h. The graph shows the ratio of the level of fluorescence per cell to that of LPS 0 ng/ml cells with or without 1 mM cystamine.
of phagocytosing cells/total cells. Representative photographs are also shown at the same sight of phase-contrast and fluorescent images above graphs (a, b, respectively). Scale bar = 25 μm. Data are means ± SD of 3 independent experiments. * p < 0.05, ** p < 0.01, vs. LPS 0 ng/ml, ## p < 0.01, vs. LPS 300 ng/ml alone.
LPS-Stimulated TG2 Expression Enhances Phagocytosis in Microglia
Neuroimmunomodulation DOI: 10.1159/000365484
b Uptake of dead cells for 30 min. The graph shows the percentage
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expression and NO production by cystamine. BV-2 cells were stimulated by 300 ng/ml LPS for 24 h in the presence or absence of 1 mM cystamine. a iNOS protein expression was detected by Western blotting. Typical bands of Western blotting for iNOS and β-actin proteins were shown in the photograph and the graph shows the iNOS/β-actin ratio. b The concentration of nitrite in the medium was measured by fluorescent assay with DAN reagent. Data are means ± SD of 3–4 samples. ** p < 0.01, vs. LPS 0 ng/ml, ## p < 0.01, vs. LPS 300 ng/ml.
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Fig. 4. Inhibition of LPS-stimulated iNOS
blocked by 1 mM cystamine (fig. 5b). When we used fluorescent large-sized (4 μm in diameter) microbeads, LPS had no apparent effect on the uptake in BV-2 cells (data not shown).
Discussion
Endocytosis is generally categorized into pinocytosis and phagocytosis depending mainly on the size of the particle taken up; pinocytosis is experimentally defined as the uptake of particles smaller than 1∼2 μm and phagocytosis as the uptake of particles over 2 μm [14–16]. In case of scavenging apoptotic cells, phagocytotic cells need to contact with ‘eat-me’ signals of target apoptotic cells by their receptors [8, 9]. PS is known to be a major ‘eat-me’ signal, most of which normally exists in the inner leaflet of the cell membrane; however, when cells undergo apoptosis, PS is exposed on the surface of the outer leaflet. In the present study, LPS stimulation increased pinocytosis of 1-μm microbeads and phagocytosis of dead cells in BV-2 cells. However, LPS did not increase the uptake of 4-μm microbeads, suggesting that BV-2 cells need an ‘eatme’ signal to phagocytose particles over 2 μm in diameter. It is reported that the phagocytosing activity was decreased in peritoneal macrophages of TG2-knockout mice and that TG2 protein interacted with adaptor proteins of PS independently of TG enzyme activity [11]. In the present study, we found that LPS-induced enhancement of endocytosis, both phagocytosis and pinocytosis, was blocked by cystamine, an inhibitor of TG enzyme activity; suggesting that TG enzyme activity is involved in microglial endocytotic mechanisms. However, TG2 protein is also reported to be involved in TG enzyme activityindependent binding of apoptotic cells as describe above. We also found in this study that cystamine simultaneously blocked LPS-induced TG2 expression as well as TG enzyme activity. Further investigations are necessary to
clarify which is more important for microglial endocytosis: TG enzyme activity or TG2 expression. Exposure to LPS has been assumed to stimulate an intracellular signaling pathway through nuclear factor (NF)-κB activation [17, 18]. In addition, the NF-κB binding site is identified on the TG2 promoter region [19]. It has been reported that in BV-2 cells TG enzyme activity is increased following LPS exposure and that NF-κB activation might be involved in TG2 expression [20]. We could confirm that LPS-stimulated TG2 mRNA expression decreased in the presence of an NF-κB inhibitor, ammonium pyrrolidinedithiocarbamate, suggesting that NF-κB is associated with LPS-induced TG2 expression (data not shown). Moreover, Park et al. [20] and Lee et al. [21] reported that inhibitors of TG enzyme activity reduced LPS-induced NO production and TG2 polymerized IκB to activate NF-κB, indicating that TG2 augments NF-κB activation in a self-enhancing manner. In the present study, LPS stimulation up-regulated TG2 expression, and cystamine inhibited the LPS-induced expression of TG2 and iNOS. It is suggested that TG enzyme activity was involved in TG2 expression via NF-κB activation. We demonstrated that TG2 expression and TG enzyme activity are closely associated with both NO production and phagocytosis in microglia. Further elucidation of the involvement of TG2 in microglial activation might help to develop a strategy against neuronal death and an approach to therapy for neurodegenerative diseases.
Acknowledgment This work was supported in part by a grant from the Japan Science and Technology Agency (Adaptable and Seamless Technology Transfer Program through target-driven R&D) to K.T. and by Grants in Aid for Scientific Research to Y.N. (24621008) and M.M. (23580408) from the Ministry of Education, Science and Culture of Japan.
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1 Kreutzberg GW: Microglia: a sensor for pathological events in the CNS. Trends Neurosci 1996;19:312–318. 2 Nakamura Y: Regulating factors for microglial activation. Biol Pharm Bull 2002;25:945–953. 3 Neniskyte U, Neher JJ, Brown GC: Neuronal death induced by nanomolar amyloid β is mediated by primary phagocytosis of neurons by microglia. J Biol Chem 2011; 286: 39904–39913.
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9 Ravichandran KS: Beginnings of a good apoptotic meal: the find-me and eat-me signaling pathways. Immunity 2011;35:445–455. 10 Szondy Z, Sarang Z, Molnár P, Németh T, Piacentini M, Mastroberardino PG, Falasca L, Aeschlimann D, Kovács J, Kiss I, Syegeydi E, Lakos G, Rajnavölgyi É, Birckbichler PJ, Melino G, Fésüs L: Tranglutaminase 2–/– mice reveal a phagocytosis-associated crosstalk between macrophages and apoptotic cells. Proc Natl Acad Sci USA 2003;100:7812–7817. 11 Tóth B, Garabuczi É, Sarang Z, Vereb G, Vámosi G, Aeschimann D, Blaskó B, Bécsi B, Erdõdi B, Lacy-Hulbert A, Zhang A, Falasca L, Birge RB, Balajthy Z, Melino G, Fésüs L, Szondy Z: Transglutaminase 2 is needed for the formation of an efficient phagocyte portal in macrophages engulfing apoptotic cells. J Immunol 2009;182:2084–2092. 12 Beck KE, De Girolamo LA, Griffin M, Billett EE: The role of tissue transglutaminase in 1-methyl-4-phenylpyridium (MPP+)-induced toxicity in differentiated human SH-SY5Y neuroblastoma cells. Neurosci Lett 2006; 11: 46–51.
Neuroimmunomodulation DOI: 10.1159/000365484
Lipopolysaccharide-Stimulated Transglutaminase 2 Expression Enhances Endocytosis Activity in the Mouse Microglial Cell Line BV-2 Kenji Kawabe Katsura Takano Mitsuaki Moriyama Yoichi Nakamura Laboratory of Integrative Physiology in Veterinary Sciences, Osaka Prefecture University, Izumisano, Japan
Abstract Objectives: In peripheral macrophages, tissue-type transglutaminase (TG2) is reported to be involved in phagocytosis of apoptotic cells. However, the contribution of TG2 to microglial phagocytosis has not been investigated. In this study, using a microglial cell line, BV-2, we examined the changes in TG2 expression, phagocytosis and pinocytosis in cells stimulated by lipopolysaccharide (LPS). Methods: Cells of the mouse microglial cell line BV-2 were stimulated by LPS with or without cystamine, an inhibitor of TG enzyme activity, for 24 h. TG2 expression was measured by real-time RTPCR and Western blotting. TG activity was evaluated using biotinylated pentylamine as a substrate. Pinocytosis was determined by uptake of 1-μm fluorescent microbeads. Phagocytosis was assessed by uptake of dead cells, human neuroblastoma SH-SY5Y cells, which were pretreated with H2O2 for 24 h. Results: Phagocytosis of dead cells and pinocytosis of fluorescent microbeads were up-regulated by LPS stimulation together with TG2 expression. Blockade of TG enzyme activity by cystamine suppressed TG2 expression, phagocytosis and pinocytosis. Conclusions: These results suggested that LPS-induced TG2 was involved in the mechanism of pinocytosis and phagocytosis in microglia. © 2014 S. Karger AG, Basel
© 2014 S. Karger AG, Basel 1021–7401/14/0000–0000$39.50/0 E-Mail [email protected] www.karger.com/nim
Introduction
Microglia categorized into a type of macrophages are considered to be a major cell for immunity in CNS. Activated microglia release nitric oxide (NO) and proinflammatory cytokines to damage neurons, and also produce neurotrophins to protect neurons. In addition, they engulf invading microorganisms and scavenge cell debris and damaged cells [1, 2]. Microglial phagocytosis contributes to the maintenance of CNS homeostasis and the development of a neural network physiologically. However, it is reported that NO production and phagocytosis of hyperactivated microglia cause neuronal death in neurodegenerative diseases [3]. Several studies found that tissue-type transglutaminase (TG2) is overexpressed in brains affected with Alzheimer’s, Parkinson’s and Huntington’s diseases [4, 5], indicating that TG2 plays an important role in CNS diseases. However, there are few reports on microglial TG2 functions. TG2 is ubiquitously expressed in various cells and shows Ca2+ concentration-dependent enzyme activity; it catalyzes protein cross-linking between glutamine and lysine residues of proteins to form ε-(γ-glutamyl)lysine isopeptide bonds [4, 5]. In addition, TG2 protein has binding domains to integrin and fibronectin in a Ca2+-independent manner [4, 5]. These functions contribute to extracellular matrix formation, tissue structure stabilization and epithelia barrier functions. Moreover, TG2 is reported to act also as a G-protein, protein disulfide isomDr. Katsura Takano Laboratory of Integrative Physiology in Veterinary Sciences, Osaka Prefecture University 1-58, Rinku-Ourai Kita Izumisano, Osaka 598-8531 (Japan) E-Mail takano @ vet.osakafu-u.ac.jp
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Key Words Cystamine · Endocytosis · Microglia · Phagocytosis · Pinocytosis · Transglutaminase 2
erase and protein kinase, mediating various intracellular signaling pathways [4–7]. These various functions of TG2 contribute to proliferation, apoptosis, migration and phagocytosis, for example [4, 5]. When phagocytes engulf dead/apoptotic cells, they need to interact with the ‘eat-me’ signal, such as phosphatidylserine (PS), which is exposed on the surface of target cells [8, 9]. It is suggested that TG2 protein in peritoneal macrophages is involved in the recognition of apoptotic cells as a result of the reduced phagocytic capacity in TG2 null macrophages [10, 11]. TG2 might be associated with microglial phagocytosis as well as peritoneal macrophages. In the present study, we examined the effect of lipopolysaccharide (LPS) stimulation on phagocytotic activity of microglia, as well as on TG2 expression and TG enzyme activity in the mouse microglial cell line BV-2.
Experimental Procedures Cell Culture Mouse microglial cell line BV-2 cells were grown and maintained in Dulbecco’s modified Eagle medium (DMEM; Gibco, Grand Island, N.Y., USA) supplemented with 10% fetal bovine serum (Nichirei, Tokyo, Japan) and penicillin/streptomycin at 37 ° C in a humidified incubator under 5% CO2. Cells were subcultured once a week. BV-2 cells were kindly provided by the Laboratory of Molecular Pharmacology, Kanazawa University Graduate School.
Real-Time RT-PCR For real-time RT-PCR, BV-2 cells (2 × 105 cells/well) were replated onto non-coating 24-well plates (Sumilon; Sumitomo Bakelite Co., Tokyo, Japan) and stimulated with LPS (from Salmonella serovar Enteritidis or Escherichia coli; Sigma, St. Louis, Mo., USA) for 24 h. Total RNA was isolated using a FavorPrepTM tissue total RNA purification mini kit (Favorgen, Ping-Tung, Taiwan, ROC). Complementary DNA was prepared using an Omniscript reverse transcription kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. PCR experiments were done using a SYBR Green real-time PCR master mix (Toyobo, Osaka, Japan) for TG2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primer sequences used were: TG2 sense strand (5′-ACT TCG ACG TGT TTG CCC ACA T-3′), TG2 antisense strand (5′-TTG ATG TCC TCA GTG CCA CAC T-3′); GAPDH sense strand (5′TTG TCA GCA ATG CAT CCT GC-3′), and GAPDH antisense strand (5′-AAT GGG AGT TGC TGT TGA AGT C-3′; Operon, Tokyo, Japan). The PCR products are 127 bp for TG2 and 194 bp for GAPDH. The conditions of each PCR cycle for these primers were as follows: denaturation at 95 ° C for 15 s; annealing at 65 ° C for 15 s, and extension at 60 ° C for 30 s. Detection of the fluorescent product was carried out at the end of the extension period. To compare mRNA levels among samples, mRNA of each gene of interest was normalized to the expression of a housekeeping gene, GAPDH, using the comparative Ct method. The changes in the levels of each PCR product were calculated as percent of control.
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TG Activity Assay Total TG activity was determined using a method developed by Beck et al. [12] with modifications. BV-2 cells (8 × 105 cells/dish) were plated onto non-coating 35-mm dishes and stimulated with 300 ng/ml LPS for 24 h in the presence of 250 μM biotinylated pentylamine (Thermo Fisher Scientific, Waltham, Mass., USA) as the substrate for TG enzyme. Cells were washed, harvested and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by membrane blotting similarly to the Western blotting assay described above. After blocking treatment, the membrane was reacted with 2.5 μg/ml NeutrAvidin-HRP (Thermo Fisher Scientific) dissolved in blocking buffer for 1 h, and TG activity was detected with HRP substrate. Protein profiles were analyzed using a lumino image analyzer (LAS-4000). The total density of 25- to 250-kDa detection bands in each lane measured by ImageJ software was evaluated. Nitrite Assay NO released from BV-2 cells was analyzed by assaying the levels of nitrite (NO2–), a relatively stable metabolite of NO. Amounts of NO2– accumulated in the medium were determined by a fluorometric method using 2,3-diaminonaphthalene (DAN) (Dojindo, Kumamoto, Japan), as described previously [13]. BV-2 cells (4 × 104 cells/well) replated onto 96-well plates were stimulated with 300 ng/ml LPS for 24 h. A 100-μl aliquot of cell-free supernatant was mixed with 20 μl of DAN solution; DAN stock solution (5 mg/ ml in 0.62 M HCl) was freshly diluted 100 times with 0.62 M HCl. The reaction was stopped with 100 μl of 0.28 M NaOH after a 10min incubation at room temperature, and the intensity of the fluorescent product, 2,3-diaminonaphthotriazole, was measured using
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Western Blotting For Western blotting, BV-2 cells (8 × 105 cells/dish) were replated onto non-coating 35-mm dishes (Sumilon) and stimulated with LPS for 24 h. Cells were homogenized in 20 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA and protease inhibitor cocktail (Sigma P8340). Each homogenate was mixed with one fourth volume of 50 mM Tris-HCl buffer (pH 6.8) containing 50% glycerol, 10% sodium dodecyl sulfate, 0.05% bromophenol blue and 25% 2-mercaptoethanol, followed by boiling at 100 ° C for 5 min. Each aliquot in a certain amount of protein was loaded onto a 10% polyacrylamide gel for electrophoresis at a constant voltage of 120 V for 2.5 h and subsequent blotting to a polyvinylidene fluoride membrane previously treated with 100% methanol. After blocking with 5% skimmed milk dissolved in 20 mM Tris-HCl buffer (pH 7.5) containing 137 mM NaCl and 0.05% Tween 20, the membrane was reacted with antibodies against TG2 (1: 500; Abcam, Cambridge, UK), induced NO synthase (iNOS; 1:1,000, from Dr. Ihara, Osaka Prefecture University, Graduate School of Science) or β-actin (1: 100,000; Sigma) followed by a reaction with anti-mouse IgG antibody (1: 10,000; Bio-Rad, Hercules, Calif., USA) conjugated with horseradish peroxidase (HRP). Proteins reactive with those antibodies were detected using ImmobilonTM Western chemiluminescent HRP substrate (Millipore, Billerica, Mass., USA). Detection bands were assessed using a lumino image analyzer (LAS-4000; Fujifilm, Tokyo, Japan). The ratios of TG2/βactin or iNOS/β-actin of detection bands were evaluated. Protein concentrations were determined by the method using CBB color solution (Nacalai Tesque, Kyoto, Japan), according to the manufacturer’s protocol, with bovine serum albumin as standard.
Data Analysis For statistical analysis of the data, one-way ANOVA followed by Tukey’s multiple comparison procedure or Student’s t test was used. Differences between treatments were considered statistically significant when p < 0.05.
Results
Up-Regulation of TG2 Expression by LPS in BV-2 Cells We examined the effect of LPS on TG2 expression in BV-2 cells. When the cells were stimulated with various concentrations of LPS for 24 h, TG2 mRNA expression increased in a concentration-dependent manner; significant increases were observed with more than 100 ng/ml LPS (fig. 1a). Protein expression of TG2 was also augmented markedly by 100 and 300 ng/ml LPS (fig. 1b). Blockade of LPS-Induced TG2 Expression and TG Activity by a TG Inhibitor We assessed the effect of cystamine, an inhibitor of TG enzyme activity, on TG2 expression. The cells were stimulated by 300 ng/ml LPS with or without 1 mM cystamine for 24 h, and then we assessed the expression levels of TG2 mRNA and protein. Cystamine significantly suppressed LPS-Stimulated TG2 Expression Enhances Phagocytosis in Microglia
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Fig. 1. Expression of TG2 in activated BV-2 cells by LPS stimula-
tion. BV-2 cells were stimulated by various concentrations of LPS for 24 h. a mRNA expression of TG2 and GAPDH was assessed by real-time RT-PCR. b TG2 protein expression was detected by Western blotting. Typical bands of Western blotting for TG2 and β-actin proteins were shown in the photograph. The graph shows the TG2/β-actin ratio of the density of detection bands. Data are means ± SD of 3 samples. * p < 0.05, ** p < 0.01, vs. LPS 0 ng/ml.
LPS-induced TG2 mRNA (fig. 2a) and protein (fig. 2b) expression. In order to confirm that cystamine actually inhibited TG enzyme activity under the conditions used, we assessed total TG activity during LPS stimulation for 24 h. TG enzyme activity increased significantly following stimulation with 300 ng/ml LPS. In the presence of 1 mM cystamine, the LPS-induced elevation in TG enzyme activity was depressed (fig. 3). Effects of Cystamine on LPS-Induced iNOS Expression and NO Production We examined whether LPS-induced TG expression and activity were related to the induction of iNOS and Neuroimmunomodulation DOI: 10.1159/000365484
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Endocytosis Assay BV-2 cells (2 × 105 cells/well) were replated onto non-coating 24-well plates and stimulated with LPS for 24 h. Pinocytosis was assessed by uptake of small-sized microbeads (diameter = 1 μm; FluoSphere®; carboxylate modified fluorescent microspheres, orange fluorescent; Molecular Probes, Eugene, Oreg., USA). After stimulation, microbeads (0.013% solid) were added to BV-2 cells and incubated for 1 h. Then BV-2 cells were washed with DMEM 3 times and fixed with 4% paraformaldehyde for fluorescent microscopy. After taking photographs, more than 20 pinocytotic cells were picked up to measure cellular fluorescence intensity by ImageJ software, and average intensity was evaluated. Phagocytosis was assessed by uptake of dead cells. Human neuroblastoma SH-SY5Y cells were treated with 200 μM H2O2 for 24 h to induce apoptosis. We confirmed that the treated cells were apoptotic exposing PS on cell surface with fluorescein isothiocyanate conjugated annexin V (Medical and Biological Laboratories, Nagoya, Japan). Apoptotic cells were stored at –80 ° C. Before use, dead cells were thawed and stained with 10 μg/ml propidium iodide for 30 min at room temperature; then the stained dead cells (2 × 106 cells/well) were added to BV-2 cells and incubated for 30 min. Then BV-2 cells were washed with DMEM three times and fixed with 4% paraformaldehyde. After taking photographs of phase-contrast and fluorescence images, total BV-2 cells and fluorescence superimposed BV-2 cells were counted.
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expression by cystamine. BV-2 cells were stimulated by 300 ng/ml LPS for 24 h in the presence or absence of 1 mM cystamine. a mRNA expression of TG2 and GAPDH was assessed by real-time RT-PCR. b TG2 protein expression was detected by Western blotting. The results are shown in the photograph and graph similar to figure 1b. Data are means ± SD of 3–4 samples. * p < 0.05, ** p < 0.01, vs. LPS 0 ng/ml, # p < 0.05, ## p < 0.01, vs. LPS 300 ng/ml.
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activities of NO production. Protein expression of iNOS was remarkably up-regulated by 300 ng/ml LPS, and 1 mM cystamine blocked LPS-induced up-regulation of iNOS expression to the basal level (fig. 4a). LPS-evoked NO production was also completely suppressed by 1 mM cystamine (fig. 4b). Effects of Cystamine on LPS-Enhanced Endocytosis We assessed two types of endocytosis activities with fluorescent small-sized microbeads (1 μm in diameter) and apoptotic/dead cells: pinocytosis and phagocytosis, respectively (for details, see Discussion). The percentages of pinocytosis action cells were about 30∼50% of total cells and not significantly different with LPS and cysta4
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tivity by cystamine. TG activity was assayed using biotinylated pentylamine (see Experimental Procedures). The photograph shows a typical protein profile produced by TG enzyme activity. The graph shows the total density of 25- to 250-kDa detection bands in each lane. Data are means ± SD of 3 samples. ** p < 0.01, vs. LPS 0 ng/ml, # p < 0.05, vs. LPS 300 ng/ml.
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mine stimulation, but pinocytosis activities of each cell changed remarkably. Fluorescence of microbeads in LPSstimulated cells increased in a concentration-dependent manner, with significant differences at concentrations of 30–300 ng/ml compared with control cells without LPS stimulation. The LPS-enhanced fluorescence was suppressed in the presence of 1 mM cystamine, and levels returned almost to the control level (fig. 5a). In control BV-2 cells, a small percentage of cells showed phagocytotic action, incorporating dead cells. However, when the cells were stimulated with 300 ng/ml LPS, more than 20% of the cells were able to incorporate dead cells. Some BV-2 cells took up more than 2 dead cells. The LPS-enhanced dead cell incorporation was Kawabe/Takano/Moriyama/Nakamura
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Fig. 2. Inhibition of LPS-stimulated TG2
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Fig. 5. Inhibition of LPS-stimulated endocytosis by cystamine. BV-2 cells were stimulated by various concentrations of LPS for 24 h in the presence or absence of 1 mM cystamine before the following uptake measurements. a Uptake of fluorescent microbeads for 1 h. The graph shows the ratio of the level of fluorescence per cell to that of LPS 0 ng/ml cells with or without 1 mM cystamine.
of phagocytosing cells/total cells. Representative photographs are also shown at the same sight of phase-contrast and fluorescent images above graphs (a, b, respectively). Scale bar = 25 μm. Data are means ± SD of 3 independent experiments. * p < 0.05, ** p < 0.01, vs. LPS 0 ng/ml, ## p < 0.01, vs. LPS 300 ng/ml alone.
LPS-Stimulated TG2 Expression Enhances Phagocytosis in Microglia
Neuroimmunomodulation DOI: 10.1159/000365484
b Uptake of dead cells for 30 min. The graph shows the percentage
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expression and NO production by cystamine. BV-2 cells were stimulated by 300 ng/ml LPS for 24 h in the presence or absence of 1 mM cystamine. a iNOS protein expression was detected by Western blotting. Typical bands of Western blotting for iNOS and β-actin proteins were shown in the photograph and the graph shows the iNOS/β-actin ratio. b The concentration of nitrite in the medium was measured by fluorescent assay with DAN reagent. Data are means ± SD of 3–4 samples. ** p < 0.01, vs. LPS 0 ng/ml, ## p < 0.01, vs. LPS 300 ng/ml.
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Fig. 4. Inhibition of LPS-stimulated iNOS
blocked by 1 mM cystamine (fig. 5b). When we used fluorescent large-sized (4 μm in diameter) microbeads, LPS had no apparent effect on the uptake in BV-2 cells (data not shown).
Discussion
Endocytosis is generally categorized into pinocytosis and phagocytosis depending mainly on the size of the particle taken up; pinocytosis is experimentally defined as the uptake of particles smaller than 1∼2 μm and phagocytosis as the uptake of particles over 2 μm [14–16]. In case of scavenging apoptotic cells, phagocytotic cells need to contact with ‘eat-me’ signals of target apoptotic cells by their receptors [8, 9]. PS is known to be a major ‘eat-me’ signal, most of which normally exists in the inner leaflet of the cell membrane; however, when cells undergo apoptosis, PS is exposed on the surface of the outer leaflet. In the present study, LPS stimulation increased pinocytosis of 1-μm microbeads and phagocytosis of dead cells in BV-2 cells. However, LPS did not increase the uptake of 4-μm microbeads, suggesting that BV-2 cells need an ‘eatme’ signal to phagocytose particles over 2 μm in diameter. It is reported that the phagocytosing activity was decreased in peritoneal macrophages of TG2-knockout mice and that TG2 protein interacted with adaptor proteins of PS independently of TG enzyme activity [11]. In the present study, we found that LPS-induced enhancement of endocytosis, both phagocytosis and pinocytosis, was blocked by cystamine, an inhibitor of TG enzyme activity; suggesting that TG enzyme activity is involved in microglial endocytotic mechanisms. However, TG2 protein is also reported to be involved in TG enzyme activityindependent binding of apoptotic cells as describe above. We also found in this study that cystamine simultaneously blocked LPS-induced TG2 expression as well as TG enzyme activity. Further investigations are necessary to
clarify which is more important for microglial endocytosis: TG enzyme activity or TG2 expression. Exposure to LPS has been assumed to stimulate an intracellular signaling pathway through nuclear factor (NF)-κB activation [17, 18]. In addition, the NF-κB binding site is identified on the TG2 promoter region [19]. It has been reported that in BV-2 cells TG enzyme activity is increased following LPS exposure and that NF-κB activation might be involved in TG2 expression [20]. We could confirm that LPS-stimulated TG2 mRNA expression decreased in the presence of an NF-κB inhibitor, ammonium pyrrolidinedithiocarbamate, suggesting that NF-κB is associated with LPS-induced TG2 expression (data not shown). Moreover, Park et al. [20] and Lee et al. [21] reported that inhibitors of TG enzyme activity reduced LPS-induced NO production and TG2 polymerized IκB to activate NF-κB, indicating that TG2 augments NF-κB activation in a self-enhancing manner. In the present study, LPS stimulation up-regulated TG2 expression, and cystamine inhibited the LPS-induced expression of TG2 and iNOS. It is suggested that TG enzyme activity was involved in TG2 expression via NF-κB activation. We demonstrated that TG2 expression and TG enzyme activity are closely associated with both NO production and phagocytosis in microglia. Further elucidation of the involvement of TG2 in microglial activation might help to develop a strategy against neuronal death and an approach to therapy for neurodegenerative diseases.
Acknowledgment This work was supported in part by a grant from the Japan Science and Technology Agency (Adaptable and Seamless Technology Transfer Program through target-driven R&D) to K.T. and by Grants in Aid for Scientific Research to Y.N. (24621008) and M.M. (23580408) from the Ministry of Education, Science and Culture of Japan.
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