Mol Neurobiol (2014) 49:1487–1500 DOI 10.1007/s12035-014-8697-6
Histamine Induces Upregulated Expression of Histamine Receptors and Increases Release of Inflammatory Mediators from Microglia Hongquan Dong & Wei Zhang & Xiaoning Zeng & Gang Hu & Huiwen Zhang & Shaoheng He & Shu Zhang
Received: 19 September 2013 / Accepted: 24 March 2014 / Published online: 22 April 2014 # Springer Science+Business Media New York 2014
Abstract Histamine is a potent mediator of inflammation and a regulator of innate and adaptive immune responses. However, the influence of histamine on microglia, the resident immune cells in the brain, remains uninvestigated. In the present study, we found that microglia can constitutively express all four histamine receptors (H1R, H2R, H3R, and H4R), and the expression of H1R and H4R can be selectively upregulated in primary cultured microglia in a dose-dependent manner by histamine. Histamine can also dose-dependently stimulate microglia activation and subsequently production of proinflammatory factors tumor necrosis factor (TNF)-alpha and interleukin-6 (IL-6). The antagonists of H1R and H4R but not H2R and H3R reduced histamine-induced TNF-alpha and IL-6 production, MAPK and PI3K/AKT pathway activation, and mitochondrial membrane potential loss in microglia, suggesting that the actions of histamine are via H1R and H4R. On the other hand, inhibitors of JNK, p38, or PI3K suppressed histamine-induced TNF-alpha and IL-6 release from microglia. Histamine also activated NF-kappa B and ammonium pyrrolidinedithiocarbamate, an inhibitor of NF-kappa B, and reduced histamine-induced TNF-alpha and IL-6 release. In Hongquan Dong and Wei Zhang contributed equally to this work. H. Dong : W. Zhang : X. Zeng : H. Zhang : S. He (*) : S. Zhang (*) Clinical Research Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, China e-mail: [email protected] e-mail: [email protected] H. Dong Department of Anesthesiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, People’s Republic of China G. Hu Department of Pharmacology, Jiangsu Key Laboratory of Neurodegeneration, Nanjing Medical University, Nanjing, People’s Republic of China
summary, the present study identifies the expression of histamine receptors on microglia. We also demonstrate that histamine induced TNF-alpha and IL-6 release from activated microglia via H1R and H4R-MAPK and PI3K/AKT-NF-kappa B signaling pathway, which will deepen the understanding of microglia-mediated neuroinflammatory symptoms of chronic neurodegenerative disease. Keywords Histamine . Microglia activation . Histamine receptors . Inflammatory factors . Neuroinflammation
Introduction Microglia, the resident immune cells in the brain, plays a pivotal role in immune surveillance of the central nervous system (CNS). Consequently, these cells are likely to play an important role in either development of protective immune responses or progression of damaging inflammation during CNS disease states [1–4]. The activated microglia carry out many of the immune effecter functions typically associated with macrophages. When subjected to abnormal stimulation, such as neurotoxins, neuronal debris, or injury, microglia become gradually activated and produce numerous inflammatory mediators including tumor necrosis factor-alpha (TNF-α), prostaglandin E2 (PGE2), interleukin-6 (IL-6), nitric oxide (NO), and reactive oxygen species (ROS). Accumulation of these proinflammatory and cytotoxic mediators is deleterious directly to neurons and subsequently induces further activation of microglia, resulting in a vicious cycle [5, 6]. Thus, inhibition of microglia activation and subsequent inflammatory process may identify novel therapeutic strategies to eliminate deleterious effects of microglia [3]. However, the regulators and related mechanisms involved in the microglia activation are not illustrated completely.
1488
Histamine is a ubiquitous mediator of diverse physiological processes including neurotransmission and brain functions, secretion of pituitary hormones, and regulation of gastrointestinal and circulatory functions [7]. Additionally, histamine is a potent mediator of inflammation and a regulator of innate and adaptive immune responses. There are four histamine receptors namely H1, H2, H3, and H4 receptors being identified. Histamine seems primarily a proinflammatory agent through H1 receptor (H1R)-mediated activities [8], whereas its physiological actions appeared to be exerted by H2 receptor (H2R). H3 receptor (H3R) contributes to gastric acid production and CNS function, respectively [9], and the H4 receptor (H4R) is the newest member of the histamine receptor family [10]. The H4R is involved in chemotaxis and inflammatory mediator release from eosinophils, mast cells, monocytes, dendritic cells, and T cells [11]. It has been discovered that brain histamine is involved in a wide range of physiological functions such as regulation of sleep–wake cycle, arousal, appetite control, cognition, learning, and memory mainly through the four subtypes of receptors H1, H2, H3, and H4. However, little is known about the role of histamine in brain microglia until very recently, a report showed that histamine inhibited LPS-induced microglia migration and release of interleukin-1beta (IL-1β) via H4R activation [12]. In the present study, we investigated the protein expression of all of the four histamine receptors on microglia and the mechanism of histamine-induced microglia activation.
Materials and Methods Reagents Dulbecco’s modified Eagle’s medium (DMEM) and fetal calf serum (FCS) were purchased from Gibco–BRL (Grand Island, NY, USA). Histamine, SP600125, SB203580, Wortmannin, 3-(4, 5-dimethylthiazol-2-yl)-2-,5-diphenyltetrazolium bromide (MTT), and mouse anti-glial fibrillary acidic protein (GFAP) antibody were purchased from Sigma– Aldrich (St. Louis, MO, USA). H 1 R agonist 2pyridylethlamine dihydrochloride (2-pyridylethlamine), H1R antagonist cetirizine dihydrochloride (cetirizine), H2R agonist amthamine dihydrobromide (amthamine), H2R antagonist ranitidine hydrochloride (ranitidine), H3R agonist (R)-(−)-αmethylhistamine dihydrobromide ((R)-(−)-αmethylhistamine), H3R antagonist carcinine ditrifluoroacetate (carcinine), H4R agonist 4-methylhistamine dihydrochloride (4-methylhistamine), and H 4 R antagonist A943931 dihydrochloride (A943931) were purchased from Tocris Bioscience (Bristol, UK). Fluoroshield mounting medium with 4,6-diamidino-2-phenylindole (DAPI) was purchased from Abcam (HK). Rat IL-6 Immunoassay Kit and Rat TNF-α Immunoassay Kit were obtained from R&D Systems, Inc.
Mol Neurobiol (2014) 49:1487–1500
(Minneapolis, MN, USA). MitoProbe™ JC-1 assay kit was purchased from Molecular Probes Invitrogen (Carlsbad, CA, USA). Specific PE-conjugated mouse anti-rat CD11b (OX42) monoclonal antibody (a marker for microglia) and isotype control antibody, specific PE-conjugated mouse anti-rat ED8 (anti-CD11b/CD18) monoclonal antibody (a marker for complement receptor 3 of activated microglia) and isotype control antibody, and mouse anti-rat ED8 (anti-CD11b/CD18) monoclonal antibody were purchased from AbD Serotec (Raleigh, NC, USA). Specific mouse monoclonal anti-OX42 antibody and rabbit monoclonal anti-H3 receptor antibody were purchased from Abcam (HK). Specific rabbit polyclonal anti-H1 receptor and rabbit polyclonal anti-H2 receptor antibodies were purchased from Alomone Labs (Ltd, Israel), and rabbit polyclonal anti-H4 receptor and anti-β-actin were purchased from Santa Cruz (CA, USA). Specific rabbit moloclonal antibodies against p38, Phospho-p38, JNK, Phospho-JNK, ERK, Phospho-ERK, AKT, Phospho-AKT, and NF-kappa B (NF-κB), mouse moloclonal antibodies against I kappa B-α (IκBα) and phosphor- IκBα, goat anti-mouse secondary antibody, and goat anti-rabbit secondary antibody were obtained from Cell Signaling (Beverly, MA, USA). PE-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG antibody were purchased from BD (BD Biosciences, USA). Preparation of Microglia-Enriched Cultures Rat primary microglia was prepared according to previously described protocol with slight modifications [13]. Briefly, tissues from whole brains of postnatal (P1–P2) Sprague– Dawley rats were triturated, and then cells were plated on poly-D-lysine precoated cell culture flasks in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % fetal calf serum, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cultures were maintained at 37 °C in a humidified atmosphere of 5 % CO2 per 95 % air. After reaching a confluent monolayer of glial cells (10–14 days), microglia were separated from astrocytes by shaking off for 5 h at 100 rpm and replated on 24-well culture plates at a density of 105 cells/cm2. The enriched microglia was >98 % pure as determined by OX-42 (CD11b)-IR. The enriched astrocytes were >96 % positive for glial fibrillary acidic protein (GFAP) as assessed by immunocytochemical staining. Challenge of Microglia Cultured microglia at a density of 1×106 cells/ml were incubated with the serum-free basal medium for 6 h before challenge. For challenge experiments, microglia was exposed to various concentrations of histamine (0.001, 0.01, 0.1, and 1 μg/ml) for 24 h or 0.1 μg/ml histamine for 0.5, 2, 6, and 24 h. For certain experiments, microglia was preincubated with 10 μM H1R antagonist cetirizine, H2R antagonist
Mol Neurobiol (2014) 49:1487–1500
ranitidine, H3R antagonist Carcinine ditrifluoroacetate, and H4R antagonist A943931 for 30 min before adding 0.1 μg/ml histamine. At 24 h following incubation, the culture supernatants were collected for ELISA analysis. And, microglia pellet were resuspended for flow cytometry analysis. For cell signaling experiments, cultured microglia at a density of 1.5×106 cells/ml was washed twice with the serum-free basal medium and then treated with 0.1 μg/ml histamine for 15, 30, 60, 120, and 240 min, respectively. Or, microglia was pretreated with 10 μM H 1 R antagonist cetirizine and H 4 R antagonist A943931 for 30 min and then exposed to histamine (0.1 μg/ml) for 15 or 60 min. Then the micrglia was collected for Western blotting. Microglia was pretreated with the inhibitors of signaling pathways SP600125 (10 μM) and SB203580 (10 μM) for 30 min before being challenged with histamine (0.1 μg/ml) for 24 h; the cell suspensions were collected for ELISA. Cell Viability Assay Cell viability was measured by 3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyl tetrazolium bromide (MTT) method. Briefly, cells were collected and seeded in 96-well plates at a density of 105cells/cm2. After incubation for 48 h, cells were exposed to fresh medium containing various concentrations of histamine (0.001, 0.01, 0.1, and 1 μg/ml) at 37 °C. After incubation for up to 24 h, 20 μL of MTT tetrazolium salt dissolved in Hank’s balanced salt solution at a final concentration of 5 mg/ml was added to each well and incubated in the CO2 incubator for 4 h. Finally, the medium was aspirated from each well and 150 μL of DMSO was added to dissolve the formazan crystals and the absorbance of each well was obtained using a Dynatech MR5000 plate counter at test and reference wavelengths of 570 and 630 nm, respectively. TNF-α and IL-6 Assay The amount of TNF-α and IL-6 in the culture medium was measured with commercial ELISA kits from R&D Systems. Western Blotting Cells were collected and homogenized in 200 ml of lysing buffer. After incubation for 20 min on ice, cell lysate was centrifuged and protein concentration in the extracts was determined by the Bradford assay. Proteins (50 μg) in cell extracts were denatured with sodium dodecyl sulfate (SDS) sample buffer and separated by 10 % SDS–polyacrylamide gel electrophoresis. Proteins were transferred to PVDF membranes (Millipore) by using a Bio-Rad miniprotein-III wet transfer unit. The membranes were incubated with 5 % BSA dissolved in Tris-buffered saline with Tween 20 (TBST) (pH 7.5, 10 mM Tris–HCl, 150 mM NaCl, and 0.1 % Tween 20) at
1489
room temperature for 1 h. This was followed by incubating the membranes with different antibodies overnight at 4 °C. The following primary antibodies were used: rabbit polyclonal anti-H1 receptor and rabbit polyclonal anti-H2 receptor (1:200); rabbit monoclonal anti-H3 receptor (1:1,000); rabbit polyclonal anti-H4 receptor (1:200); and rabbit monoclonal anti-c-Jun N-terminal kinase (JNK), -phospho-JNK, -p38, phospho-p38, -ERK, phospho-ERK, -AKT, -phospho-AKT, IκBα, phospho-IκBα (1:1,000). After adding the goat-antrabbit secondary antibody (1:1,000) for 1 h, the protein bands on the membranes were detected with an enhanced chemiluminescence kit. Immunofluorescence To determine the expression of histamine receptors on microglia, colabeling microglia OX-42 with the H1R, H2R, H3R, and H4R was performed by dual-florescence immunostaining. Cells were fixed with 4 % paraformaldehyde for 30 min; unspecific binding was blocked by incubating cells in a 5 % BSA and 0.1 % Triton X-100 solution for 1 h at room temperature. Microglia was incubated with mouse anti-OX42 monoclonal antibody (1:200) along with rabbit polyclonal anti-H1R, anti-H2R, anti-H3R, and anti-H4R antibodies in the blocking solution overnight at 4 °C. After three washes with PBS, microglia was incubated with corresponding PEconjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG (1:200) for 2 h at room temperature. After three washes in PBS, cells were smeared on glass slides and cover slips were sealed with nail polish. To determine the effect if histamine on microglia activation, activated microglia was detected with monoclonal antibody ED8 (1:300), which recognizes complement receptor 3 (CD11b/CD18), overnight at 4 °C. After washing, microglia was incubated with PEconjugated secondary antibody (1:200) and nuclei were stained with DAPI. To detect the intracellular location of the NF-κB p65 subunit, microglia was incubated with rabbit antiNF-κB monoclonal antibody (1:1,000) overnight at 4 °C followed by FITC-conjugated goat anti-rabbit secondary antibody (1:200) and nuclei were stained with DAPI. Fluorescent images were acquired by using a confocal microscope. Flow Cytometry Analysis Microglia were pelleted by centrifugation at 450g for 10 min, and then fixed in 4 % paraformaldehyde for 30 min. After washing, the cells were re-suspended in PBS. For H1R, H2R, H3R, and H4R staining, cells were incubated with rabbit antiH1R, anti-H2R, anti-H3R, and anti-H4R antibodies or normal rabbit IgG, respectively, overnight at 4 °C, followed by 1 μg/ml of FITC-conjugated goat anti-rabbit secondary antibody alone with PE-conjugated mouse anti-rat OX-42 monoclonal antibody or isotype control (1:200) at 37 °C for 1 h. To
1490
determine the effect if histamine on microglia activation, microglia was incubated with PE-conjugated mouse anti-rat ED8 monoclonal antibody or isotype control (1:200) at 37 °C for 1 h. Cells were finally resuspended in PBS and analyzed on a FACS Calibur flow cytometer with CellQuest software (BD Biosciences, USA). Measurement of Microglia Mitochondrial Membrane Potential Microglia mitochondrial membrane potential (ΔΨm) was assessed with the MitoProbeTM JC-1 assay kit. JC-1, a cationic dye, exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (∼529 nm) to red (∼590 nm). Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. The potential-sensitive color shift is due to concentration dependent formation of red fluorescent Jaggregates. Microglia, suspended in 1 ml PBS at approximately 1×106 cells/ml, was incubated with 2 μM of JC-1 at 37 °C for 15 min, and was analyzed on a flow cytometer with 488 nm excitation using emission filters appropriate for Alexa Fluor 488 dye and R-phycoerythrin. A decrease in the ratio of red/ green fluorescence intensity was interpreted as loss of ΔΨm, whereas an increase in the ratio was interpreted as gain in ΔΨm. Statistical Analysis Values shown are means±s.e.m. The significance of the difference between control and samples treated with various compounds was determined by one-way ANOVA followed by the post hoc least significant difference test. Differences were considered significant at P
Histamine Induces Upregulated Expression of Histamine Receptors and Increases Release of Inflammatory Mediators from Microglia Hongquan Dong & Wei Zhang & Xiaoning Zeng & Gang Hu & Huiwen Zhang & Shaoheng He & Shu Zhang
Received: 19 September 2013 / Accepted: 24 March 2014 / Published online: 22 April 2014 # Springer Science+Business Media New York 2014
Abstract Histamine is a potent mediator of inflammation and a regulator of innate and adaptive immune responses. However, the influence of histamine on microglia, the resident immune cells in the brain, remains uninvestigated. In the present study, we found that microglia can constitutively express all four histamine receptors (H1R, H2R, H3R, and H4R), and the expression of H1R and H4R can be selectively upregulated in primary cultured microglia in a dose-dependent manner by histamine. Histamine can also dose-dependently stimulate microglia activation and subsequently production of proinflammatory factors tumor necrosis factor (TNF)-alpha and interleukin-6 (IL-6). The antagonists of H1R and H4R but not H2R and H3R reduced histamine-induced TNF-alpha and IL-6 production, MAPK and PI3K/AKT pathway activation, and mitochondrial membrane potential loss in microglia, suggesting that the actions of histamine are via H1R and H4R. On the other hand, inhibitors of JNK, p38, or PI3K suppressed histamine-induced TNF-alpha and IL-6 release from microglia. Histamine also activated NF-kappa B and ammonium pyrrolidinedithiocarbamate, an inhibitor of NF-kappa B, and reduced histamine-induced TNF-alpha and IL-6 release. In Hongquan Dong and Wei Zhang contributed equally to this work. H. Dong : W. Zhang : X. Zeng : H. Zhang : S. He (*) : S. Zhang (*) Clinical Research Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, China e-mail: [email protected] e-mail: [email protected] H. Dong Department of Anesthesiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, People’s Republic of China G. Hu Department of Pharmacology, Jiangsu Key Laboratory of Neurodegeneration, Nanjing Medical University, Nanjing, People’s Republic of China
summary, the present study identifies the expression of histamine receptors on microglia. We also demonstrate that histamine induced TNF-alpha and IL-6 release from activated microglia via H1R and H4R-MAPK and PI3K/AKT-NF-kappa B signaling pathway, which will deepen the understanding of microglia-mediated neuroinflammatory symptoms of chronic neurodegenerative disease. Keywords Histamine . Microglia activation . Histamine receptors . Inflammatory factors . Neuroinflammation
Introduction Microglia, the resident immune cells in the brain, plays a pivotal role in immune surveillance of the central nervous system (CNS). Consequently, these cells are likely to play an important role in either development of protective immune responses or progression of damaging inflammation during CNS disease states [1–4]. The activated microglia carry out many of the immune effecter functions typically associated with macrophages. When subjected to abnormal stimulation, such as neurotoxins, neuronal debris, or injury, microglia become gradually activated and produce numerous inflammatory mediators including tumor necrosis factor-alpha (TNF-α), prostaglandin E2 (PGE2), interleukin-6 (IL-6), nitric oxide (NO), and reactive oxygen species (ROS). Accumulation of these proinflammatory and cytotoxic mediators is deleterious directly to neurons and subsequently induces further activation of microglia, resulting in a vicious cycle [5, 6]. Thus, inhibition of microglia activation and subsequent inflammatory process may identify novel therapeutic strategies to eliminate deleterious effects of microglia [3]. However, the regulators and related mechanisms involved in the microglia activation are not illustrated completely.
1488
Histamine is a ubiquitous mediator of diverse physiological processes including neurotransmission and brain functions, secretion of pituitary hormones, and regulation of gastrointestinal and circulatory functions [7]. Additionally, histamine is a potent mediator of inflammation and a regulator of innate and adaptive immune responses. There are four histamine receptors namely H1, H2, H3, and H4 receptors being identified. Histamine seems primarily a proinflammatory agent through H1 receptor (H1R)-mediated activities [8], whereas its physiological actions appeared to be exerted by H2 receptor (H2R). H3 receptor (H3R) contributes to gastric acid production and CNS function, respectively [9], and the H4 receptor (H4R) is the newest member of the histamine receptor family [10]. The H4R is involved in chemotaxis and inflammatory mediator release from eosinophils, mast cells, monocytes, dendritic cells, and T cells [11]. It has been discovered that brain histamine is involved in a wide range of physiological functions such as regulation of sleep–wake cycle, arousal, appetite control, cognition, learning, and memory mainly through the four subtypes of receptors H1, H2, H3, and H4. However, little is known about the role of histamine in brain microglia until very recently, a report showed that histamine inhibited LPS-induced microglia migration and release of interleukin-1beta (IL-1β) via H4R activation [12]. In the present study, we investigated the protein expression of all of the four histamine receptors on microglia and the mechanism of histamine-induced microglia activation.
Materials and Methods Reagents Dulbecco’s modified Eagle’s medium (DMEM) and fetal calf serum (FCS) were purchased from Gibco–BRL (Grand Island, NY, USA). Histamine, SP600125, SB203580, Wortmannin, 3-(4, 5-dimethylthiazol-2-yl)-2-,5-diphenyltetrazolium bromide (MTT), and mouse anti-glial fibrillary acidic protein (GFAP) antibody were purchased from Sigma– Aldrich (St. Louis, MO, USA). H 1 R agonist 2pyridylethlamine dihydrochloride (2-pyridylethlamine), H1R antagonist cetirizine dihydrochloride (cetirizine), H2R agonist amthamine dihydrobromide (amthamine), H2R antagonist ranitidine hydrochloride (ranitidine), H3R agonist (R)-(−)-αmethylhistamine dihydrobromide ((R)-(−)-αmethylhistamine), H3R antagonist carcinine ditrifluoroacetate (carcinine), H4R agonist 4-methylhistamine dihydrochloride (4-methylhistamine), and H 4 R antagonist A943931 dihydrochloride (A943931) were purchased from Tocris Bioscience (Bristol, UK). Fluoroshield mounting medium with 4,6-diamidino-2-phenylindole (DAPI) was purchased from Abcam (HK). Rat IL-6 Immunoassay Kit and Rat TNF-α Immunoassay Kit were obtained from R&D Systems, Inc.
Mol Neurobiol (2014) 49:1487–1500
(Minneapolis, MN, USA). MitoProbe™ JC-1 assay kit was purchased from Molecular Probes Invitrogen (Carlsbad, CA, USA). Specific PE-conjugated mouse anti-rat CD11b (OX42) monoclonal antibody (a marker for microglia) and isotype control antibody, specific PE-conjugated mouse anti-rat ED8 (anti-CD11b/CD18) monoclonal antibody (a marker for complement receptor 3 of activated microglia) and isotype control antibody, and mouse anti-rat ED8 (anti-CD11b/CD18) monoclonal antibody were purchased from AbD Serotec (Raleigh, NC, USA). Specific mouse monoclonal anti-OX42 antibody and rabbit monoclonal anti-H3 receptor antibody were purchased from Abcam (HK). Specific rabbit polyclonal anti-H1 receptor and rabbit polyclonal anti-H2 receptor antibodies were purchased from Alomone Labs (Ltd, Israel), and rabbit polyclonal anti-H4 receptor and anti-β-actin were purchased from Santa Cruz (CA, USA). Specific rabbit moloclonal antibodies against p38, Phospho-p38, JNK, Phospho-JNK, ERK, Phospho-ERK, AKT, Phospho-AKT, and NF-kappa B (NF-κB), mouse moloclonal antibodies against I kappa B-α (IκBα) and phosphor- IκBα, goat anti-mouse secondary antibody, and goat anti-rabbit secondary antibody were obtained from Cell Signaling (Beverly, MA, USA). PE-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG antibody were purchased from BD (BD Biosciences, USA). Preparation of Microglia-Enriched Cultures Rat primary microglia was prepared according to previously described protocol with slight modifications [13]. Briefly, tissues from whole brains of postnatal (P1–P2) Sprague– Dawley rats were triturated, and then cells were plated on poly-D-lysine precoated cell culture flasks in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % fetal calf serum, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cultures were maintained at 37 °C in a humidified atmosphere of 5 % CO2 per 95 % air. After reaching a confluent monolayer of glial cells (10–14 days), microglia were separated from astrocytes by shaking off for 5 h at 100 rpm and replated on 24-well culture plates at a density of 105 cells/cm2. The enriched microglia was >98 % pure as determined by OX-42 (CD11b)-IR. The enriched astrocytes were >96 % positive for glial fibrillary acidic protein (GFAP) as assessed by immunocytochemical staining. Challenge of Microglia Cultured microglia at a density of 1×106 cells/ml were incubated with the serum-free basal medium for 6 h before challenge. For challenge experiments, microglia was exposed to various concentrations of histamine (0.001, 0.01, 0.1, and 1 μg/ml) for 24 h or 0.1 μg/ml histamine for 0.5, 2, 6, and 24 h. For certain experiments, microglia was preincubated with 10 μM H1R antagonist cetirizine, H2R antagonist
Mol Neurobiol (2014) 49:1487–1500
ranitidine, H3R antagonist Carcinine ditrifluoroacetate, and H4R antagonist A943931 for 30 min before adding 0.1 μg/ml histamine. At 24 h following incubation, the culture supernatants were collected for ELISA analysis. And, microglia pellet were resuspended for flow cytometry analysis. For cell signaling experiments, cultured microglia at a density of 1.5×106 cells/ml was washed twice with the serum-free basal medium and then treated with 0.1 μg/ml histamine for 15, 30, 60, 120, and 240 min, respectively. Or, microglia was pretreated with 10 μM H 1 R antagonist cetirizine and H 4 R antagonist A943931 for 30 min and then exposed to histamine (0.1 μg/ml) for 15 or 60 min. Then the micrglia was collected for Western blotting. Microglia was pretreated with the inhibitors of signaling pathways SP600125 (10 μM) and SB203580 (10 μM) for 30 min before being challenged with histamine (0.1 μg/ml) for 24 h; the cell suspensions were collected for ELISA. Cell Viability Assay Cell viability was measured by 3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyl tetrazolium bromide (MTT) method. Briefly, cells were collected and seeded in 96-well plates at a density of 105cells/cm2. After incubation for 48 h, cells were exposed to fresh medium containing various concentrations of histamine (0.001, 0.01, 0.1, and 1 μg/ml) at 37 °C. After incubation for up to 24 h, 20 μL of MTT tetrazolium salt dissolved in Hank’s balanced salt solution at a final concentration of 5 mg/ml was added to each well and incubated in the CO2 incubator for 4 h. Finally, the medium was aspirated from each well and 150 μL of DMSO was added to dissolve the formazan crystals and the absorbance of each well was obtained using a Dynatech MR5000 plate counter at test and reference wavelengths of 570 and 630 nm, respectively. TNF-α and IL-6 Assay The amount of TNF-α and IL-6 in the culture medium was measured with commercial ELISA kits from R&D Systems. Western Blotting Cells were collected and homogenized in 200 ml of lysing buffer. After incubation for 20 min on ice, cell lysate was centrifuged and protein concentration in the extracts was determined by the Bradford assay. Proteins (50 μg) in cell extracts were denatured with sodium dodecyl sulfate (SDS) sample buffer and separated by 10 % SDS–polyacrylamide gel electrophoresis. Proteins were transferred to PVDF membranes (Millipore) by using a Bio-Rad miniprotein-III wet transfer unit. The membranes were incubated with 5 % BSA dissolved in Tris-buffered saline with Tween 20 (TBST) (pH 7.5, 10 mM Tris–HCl, 150 mM NaCl, and 0.1 % Tween 20) at
1489
room temperature for 1 h. This was followed by incubating the membranes with different antibodies overnight at 4 °C. The following primary antibodies were used: rabbit polyclonal anti-H1 receptor and rabbit polyclonal anti-H2 receptor (1:200); rabbit monoclonal anti-H3 receptor (1:1,000); rabbit polyclonal anti-H4 receptor (1:200); and rabbit monoclonal anti-c-Jun N-terminal kinase (JNK), -phospho-JNK, -p38, phospho-p38, -ERK, phospho-ERK, -AKT, -phospho-AKT, IκBα, phospho-IκBα (1:1,000). After adding the goat-antrabbit secondary antibody (1:1,000) for 1 h, the protein bands on the membranes were detected with an enhanced chemiluminescence kit. Immunofluorescence To determine the expression of histamine receptors on microglia, colabeling microglia OX-42 with the H1R, H2R, H3R, and H4R was performed by dual-florescence immunostaining. Cells were fixed with 4 % paraformaldehyde for 30 min; unspecific binding was blocked by incubating cells in a 5 % BSA and 0.1 % Triton X-100 solution for 1 h at room temperature. Microglia was incubated with mouse anti-OX42 monoclonal antibody (1:200) along with rabbit polyclonal anti-H1R, anti-H2R, anti-H3R, and anti-H4R antibodies in the blocking solution overnight at 4 °C. After three washes with PBS, microglia was incubated with corresponding PEconjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG (1:200) for 2 h at room temperature. After three washes in PBS, cells were smeared on glass slides and cover slips were sealed with nail polish. To determine the effect if histamine on microglia activation, activated microglia was detected with monoclonal antibody ED8 (1:300), which recognizes complement receptor 3 (CD11b/CD18), overnight at 4 °C. After washing, microglia was incubated with PEconjugated secondary antibody (1:200) and nuclei were stained with DAPI. To detect the intracellular location of the NF-κB p65 subunit, microglia was incubated with rabbit antiNF-κB monoclonal antibody (1:1,000) overnight at 4 °C followed by FITC-conjugated goat anti-rabbit secondary antibody (1:200) and nuclei were stained with DAPI. Fluorescent images were acquired by using a confocal microscope. Flow Cytometry Analysis Microglia were pelleted by centrifugation at 450g for 10 min, and then fixed in 4 % paraformaldehyde for 30 min. After washing, the cells were re-suspended in PBS. For H1R, H2R, H3R, and H4R staining, cells were incubated with rabbit antiH1R, anti-H2R, anti-H3R, and anti-H4R antibodies or normal rabbit IgG, respectively, overnight at 4 °C, followed by 1 μg/ml of FITC-conjugated goat anti-rabbit secondary antibody alone with PE-conjugated mouse anti-rat OX-42 monoclonal antibody or isotype control (1:200) at 37 °C for 1 h. To
1490
determine the effect if histamine on microglia activation, microglia was incubated with PE-conjugated mouse anti-rat ED8 monoclonal antibody or isotype control (1:200) at 37 °C for 1 h. Cells were finally resuspended in PBS and analyzed on a FACS Calibur flow cytometer with CellQuest software (BD Biosciences, USA). Measurement of Microglia Mitochondrial Membrane Potential Microglia mitochondrial membrane potential (ΔΨm) was assessed with the MitoProbeTM JC-1 assay kit. JC-1, a cationic dye, exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (∼529 nm) to red (∼590 nm). Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. The potential-sensitive color shift is due to concentration dependent formation of red fluorescent Jaggregates. Microglia, suspended in 1 ml PBS at approximately 1×106 cells/ml, was incubated with 2 μM of JC-1 at 37 °C for 15 min, and was analyzed on a flow cytometer with 488 nm excitation using emission filters appropriate for Alexa Fluor 488 dye and R-phycoerythrin. A decrease in the ratio of red/ green fluorescence intensity was interpreted as loss of ΔΨm, whereas an increase in the ratio was interpreted as gain in ΔΨm. Statistical Analysis Values shown are means±s.e.m. The significance of the difference between control and samples treated with various compounds was determined by one-way ANOVA followed by the post hoc least significant difference test. Differences were considered significant at P
