Background
The co-stimulatory receptor, CD40 molecule, is a 50-kDa type I member of the tumor necrosis factor receptor superfamily that is widely expressed by the various immune and non-immune cells [
1‐
7]. The interaction between CD40 and its ligand, CD40L (CD154), is one of multiple signals necessary for a productive immune response [
8‐
10]. The CD40-CD154 interaction promotes a wide spectrum of molecular and cellular processes including, immunoglobulin class switching, cell differentiation and maturation, B-cell growth, and expression of other co-stimulatory molecules such as MHC class II, ICAM-1, VCAM-1, E-selectin, LFA-3, B7.1, and B7.2) [
11,
12]. In addition, CD40-CD154 interaction induces the production of cytotoxic radicals and of various pro-inflammatory cytokines (TNF-α, IL6, IL-8, and IL-12) and chemokines (CCL-2) [
13,
14].
In the central nervous system (CNS), the microglial cells are constantly in motion, surveying their environment to protect the nervous system acting as debris scavengers, killers of pathogens, and regulators of innate and adaptive immune responses. The microglia cells express the key surface molecules for antigen presentation (CD40, MHC-II, and B7); therefore, they are considered the most potent endogenous antigen-presenting cells in the CNS [
15]. In a healthy nervous system, microglia constitutively expresses CD40 at a low level, which is enhanced under inflammatory conditions. Several studies show that the aberrant expression of CD40 is involved in the initiation and maintenance of various neurodegenerative diseases including multiple sclerosis, Alzheimer’s disease, HIV-1-associated dementia and cerebral ischemia [
16‐
20], and other diseases as rheumatoid arthritis and atherosclerosis [
18,
21,
22]. Blockade of CD40-CD40L signaling has been shown to provide a significant beneficial effect in a number of animal models of neurological human diseases [
1,
18,
23‐
28].
Previous findings suggested that cPLA
2α plays an important role in inflammation. cPLA
2α specifically hydrolyzes phospholipids containing arachidonic acid at the sn-2 position [
29,
30] and is generally thought to be the rate-limiting step in the generation of eicosanoids and platelet activating factor. These lipid mediators play critical roles in the initiation and modulation of inflammation and oxidative stress. cPLA
2α is ubiquitous in the brain cells and is essential for their physiological regulation. However, elevated cPLA
2α expression and activity were detected in the inflammatory sites in a vast array of inflammatory diseases [
31], including neurodegenerative diseases such as Alzheimer’s disease, multiple sclerosis, and amyotrophic lateral sclerosis (ALS) [
32‐
35]. Our previous study [
36] in a mouse model of ALS, hmSOD1 G93A, demonstrated that the blunting cPLA
2α protein expression and inhibition of its activity inhibited microglial-CD40 upregulation. This inhibitory effect could be a result of a direct regulatory role of cPLA
2α on CD40 inductive process or an indirect effect due to damping of inflammation. The present study was designed to determine whether cPLA
2α has a direct role in the events leading to CD40 protein induction. To this aim, we used mouse microglia cultures and two different stimuli, LPS and IFNγ that have been reported to induce CD40 upregulation. The signal transduction events leading to CD40 upregulation by both stimuli have been studied, and it was reported that they include two transcription factors NF-κB and STAT1α that are activated in different rank order and time scale by the two stimuli [
37‐
39].
Methods
Materials
Glutamine, penicillin-streptomycin-nystatin, phosphate buffered saline (PBS) Dulbecco’s Modified Eagle’s Medium (DMEM), Hanks’ Balanced Salts Solution (HBSS), fetal bovine serum (FBS), HEPES, sodium pyruvate, Dulbecco’s Modified Eagle’s/F12 (HAM) medium (DMEM/F12) were from Beth Ha-Emek, Biological Industries, Israel.
Sodium azide, trypan blue, p-nitrophenylphosphate, phenylmethylsulfonyl fluoride, leupeptin, benzamidine, aprotinin, DMSO, Tween 20, Tris, 4,6-diamidino-2-phenylindole (DAPI), bovine serum albumin (BSA), Trypsin-EDTA, dihyroethidium (DHE), lipopolysaccharide (LPS), Skim Milk Powder, Poly-L-lysine, horseradish peroxidase (HRP), 1,2-Dioleoyl-sn-glycerol, Triton X-100, β-mercaptoethanol, Percoll, non-essential amino-acids, Diphenyliodonium chloride (DPI) were from Sigma Israel, Rehovot, Israel. Fetal calf serum was from GE Healthcare Life Sciences HyClone Laboratories, Inc., Logan Utah, USA. ECL detection kit for the immunoblot analysis was from PerkinElmer, MA, USA. Pyrrophenone was from Cayman Chemical, Michigan, USA. TNF-α-neutralizing antibody and U0126 (MEK1/2 inhibitor) were from Cell Signaling Technology, Danvers, MA, USA. Interleukin (IL)-4, IL-10, TNF-α, IFN-γ were from PeproTech Asia, NJ, USA.
Primary microglial cell culture
Microglia were isolated from the brains of mice C57BL 1-day-old pups as previously described [
40] with minor modifications. Briefly, the pups were decapitated and the brains were taken out. The tissues were digested by incubation with an enzymatic solution containing papain (116 mM NaCl, 5.4 mM KCl, 26 mM NaHCO3, 1 mM NaH
2PO
4, 1.5 mM CaCl
2, 1 mM MgSO
4, 0.5 mM EDTA, 25 mM glucose, 1 mM cysteine, and 20 U/ml papain) for 60 min at 37 °C, 5% CO
2. The enzymatic solution was quenched with 20% FBS in HBSS and centrifuged for 4 min at ×200
g. A second digestion procedure was performed by treating the brain tissues with 0.5 mg/ml DNase-I (Worthington Biochemical Corp., NJ, USA) for 5 min and gently passing it through a fire-polished Pasteur pipettes several times. Then, the digested tissues were filtered through a 70 micron cell strainer (Corning, NY, USA) and centrifuged at 200
g for 4 min. The pellet was resuspended in 20% isotonic percoll in HBSS. Fresh HBSS was carefully added and then the tubes were centrifuged at ×200
g for 20 min with slow acceleration and no brakes. The pellet containing the mixed glial cells were washed with HBSS, centrifuged at ×200
g for 4 min and then suspended in DMEM-F12 medium (10% FCS, 1% non-essential amino-acids, 11.4 μm β-mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 12.5 U/ml nystatin). The cells were seeded into Poly-L-lysine coated flasks and kept at 37 °C in a humidified atmosphere of 5% CO
2. The growth medium was replaced with a fresh after 4 days. After two weeks, the microglial cells were separated from the astroglial cell monolayer by shaking the flasks for 1 h at 120 rpm on a rotator shaker and subjected to mild trypsinization with DMEM containing 0.25% Trypsin-EDTA (1:3) for about 90 min at 37 °C and then exchange with fresh DMEM. Then, the isolated microglial cultures were treated with 0.25% Trypsin for approximately 15 min at 37 °C and carefully detached. The cells were suspended with DMEM-F12 (containing 2% FBS 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 12.5 U/ml Nystatin) and cultured (6 × 10
5 cells/ml) in 24 wells on cover-slips coated with Poly-L-lysine at 37 °C in a humidified atmosphere of 5% CO
2 for a week before the experiment. The purity of microglial cell preparations was confirmed by testing their immunoreactivity to the Iba-1 (Wako Chemicals, Richmond, VA, USA) marker.
Cell cultures
BV2 immortalized murine microglial cell line was a kind gift from Prof. Rosario Donato (Department of Biochemical Sciences, University of Perugia, Italy). The cells were maintained in DMEM containing 5% FBS 2 mM L-glutamine, 100u/ml penicillin, 100 μg/ml streptomycin, and 12.5 U/ml Nystatin at 37 °C and 5% CO2 until they reached confluence. The cells (3.5 × 105 cells/ml) were suspended in DMEM containing 2% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 12.5 U/ml Nystatin and seeded in plates of 24 or 6 wells at 37 °C in a humidified atmosphere of 5% CO2.
Flow cytometry
The microglial cells were suspended in PBS and counted by Trypan Blue. The cells were pre-incubated with rat anti-mouse Fc Blocker (BD Pharmingen, San Jose, CA) at 4 °C for 10 min. For detection of CD40, the cells were incubated with PE anti-mouse CD40 (BioLegend, San Diego, CA) for 2 h on ice in the presence of Fc Blocker. Next, the cells were washed three times with PBS and subjected to fluorescence-activated cell sorter (FACS FC 500, Switzerland, Beckman Coulter) analysis. The median (median of fluorescence intensity) was calculated by subtracting the non-specific fluorescence.
Immunofluorescence analysis
Microglia were suspended in DMEM (2% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 12.5 U/ml Nystatin) and seeded on cover slips. The cells were fixed with ice-cold methanol for 3 min and then washed with HBSS. For immunofluorescence detection, the fixed microglial cells were incubated with the first antibody 1:50 in 5% BSA/PBS (anti cPLA2α (Santa Cruz Biotechnology, CA, USA), anti CD40 (Serotec, Cambridge, UK), anti CD206 (R&D Systems, Minneapolis, USA) Serotec, Oxfordshire, UK) for 90 min at room temperature. The cells were washed three times in HBSS and incubated with Cy3 anti-rabbit, DyLight anti-rabbit, and Cy3 anti-goat (1:50 in 5% BSA/PBS; Jackson ImmunoResearch Laboratories, Inc., PA, USA) for 60 min at room temperature. The cells were washed three times in HBSS, and the nuclei were stained with DAPI. Then, final wash was performed and the cells were taken to fluorescence microscope analysis (Olympus, BX60, Hamburg, Germany).
Intracellular superoxide anion assay
O2
− production was measured using dihyroethidium (DHE). The cells were incubated in a 24-well plate on cover slips for 24 h at 37 °C. The next day the medium was replaced with heated HBSS containing 10 μm DHE, and the cells were incubated for 45 min at 37 °C. Then, the cells were stimulated with IFN-γ or LPS for 15 min. Then, the cells were stained with DAPI, washed, and fixed with ice-cold methanol for 3 min. the fluorescence intensity was measured by fluorescence microscope (Olympus, BX60, Hamburg, Germany).
Inhibition of cPLA2α expression using antisense oligonucleotides
An oligodeoxy-nucleotide antisense (
tcaaaggtctcattcc
aca) and its corresponding sense with phosphorothioate modifications on the last three bases at both 5′ and 3′ ends were used as described in our previous article [
35]. The specificity to cPLA
2α was analyzed by blast search program and was demonstrated in our previous study [
31].
Immunoblot analysis
Microglial cell lysates were prepared using lysis buffer containing: 2% Triton X-100, 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 10 μm MgCl2, 10 μg/ml leupeptin, 1 mM phenylmethylsulphonylfluoride, 10 μg/ml aprotonin, 1 mM benzamidine, 20 mM para-nitrophenyl phosphate, 5 mM sodium orthovanadate, 10 mM sodium fluoride, and 50 mM β-glycerophosphate). Cell lysates were analyzed by SDS-PAGE on 9% gels. The amount of protein in each sample was quantified with the Pierce BCA Proteins Assay using BSA standards. The resolved proteins were transferred to nitrocellulose and blocked in 5% BSA in TBS-T (10 mM Tris, 135 mM NaCl, pH 7.4, 0.1% Tween 20). The blots were incubated overnight at 4 °C with primary antibodies (anti-cPLA2α and anti-phospho-(serine-505)-cPLA2α from Sigma, anti-NF-κB p65, anti-phospho-(serine-536)-NF-κB p65, anti phopho-p44/42 ERK1/2 (Thr202/Tyr204), anti-p44/42 ERK1/2, anti-STAT1α, anti-phospho-(serine-727)-STAT1α, anti-phospho-(Thr-701)-STAT1α from Cell Signaling, MA, USA; washed and incubated with peroxidase-conjugated secondary antibodies (Amersham Pharmacia Biotech, NJ, USA) for 1.5 h at room temperature. Detection of immunoreactive bands was carried out using enhanced chemiluminescence. Changes in protein expression or phosphorylation were quantified by densitometry using ImageJ program. The intensity of each band was divided by the intensity of each total protein band and expressed as arbitrary units. The quantitative measurements are adequate to determine the changes of each protein in the same immunoblot.
Separation of plasma membranes and immunoprecipitation
Plasma membranes were separated as described before ([
41]). Cell 10
8/ml suspended in relaxation buffer (100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, 1.25 mM EGTA, 1 mM ATP, 10 mM PIPES, pH7.4) containing 1 mM PMSF and 100 μm leupeptin at 4 °C and sonicated , resulting in 95% cell breakage. After centrifugation (5 min; ×15,600
g) to remove the granules, nuclei, and unbroken cells, the supernatant was centrifuge in a Beckman Airfuge (Beckman Instrument, Fulletron, CA) 30 min; ×134,000
g to obtain cell membrane pellet and cytosol supernatant. The membranes were suspended at 10
9 cell equivalent/ml in 0.34 sucrose/half-strength relaxation buffer. The microglial cell membranes subjected to immunoprecipitation with goat anti-serum raised against recombinant p47
phox (gift from Dr. T Leto, NIAID, NIH, Bethesda, USA). Immunoprecipitation was at a final volume of 0.5 ml at 4 °C. Recombinant protein A–Sepharose beads (Zymed Laboratories Inc., CA, USA) were added to each sample, and the samples were tumbled end-over-end for 1 h. The beads were then washed six times with lysis buffer boiled in lamely buffer and subjected to SDS-PAGE analysis.
TNF-a detection–using mouse TNF-α high sensitivity ELISA, eBioscience, Vienna, Austria.
Statistical analysis
Significant differences between the parameters evaluated were determined by ANOVA using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA) followed by multiple comparisons Bonferroni post hoc correction. p value less than 0.05 were considered statistically significant.
Discussion
The present study shows that cPLA
2α is involved in the induction of CD40 by either LPS or IFNγ. Reduction of cPLA
2α upregulation by a specific antisense or inhibition of cPLA
2α activity by a specific inhibitor prevented the induction of CD40 protein expression by either LPS or IFNγ. The results suggest that cPLA
2α has a direct role in CD40 upregulation, a feature of the pro-inflammatory M1-phenotype. In accordance with this view, the regulatory role of cPLA
2α in the induction of several characters of M1 phenotype in microglia and macrophages, such as iNOS, COX2, NOX2-NADPH oxidase as well as production of eicosanoids and pro-inflammatory mediators, was reported by us and others [
40,
44]. cPLA
2α, however, is not involved in the transformation to M2-phenotype, as its protein level was not elevated by addition of IL4 + IL10, and the presence of AS did not affect the significant induction of CD206 or of arginase 1 in microglia. In accordance with our results, it was reported that the antiinflammatory cytokines IL4 or IL10 by themselves did not affect cPLA
2α activation or biosynthesis [
45,
46], further supporting the role of cPLA
2α in inflammatory processes.
The results of the present study show that superoxides generated by NOX2-NADPH oxidase participate in upregulation of CD40 expression induced by LPS or IFNγ in microglia, since inhibition of NOX-2 NADPH oxidase prevented the induction of CD40. We show here that in BV-2 microglia cell line, inhibition of the activation of cPLA
2α induced by either LPS or IFNγ, as demonstrated by the use of antisense against cPLA
2α or the specific inhibitor of cPLA
2α activity, pyrrophenone, inhibited the production of superoxides by the NOX2-NADPH oxidase. Inhibition of the oxidase activity did not affect cPLA
2α activation detected by its phosphorylated form. These results suggest that the NOX2-NADPH oxidase is regulated by cPLA
2α in microglia stimulated with either LPS or IFNγ, that is similar to ours and other studies related to the various phagocytic cells stimulated with a variety of agonists [
31,
40,
44,
47‐
50]. We show here that phoshpo-cPLA
2α translocated to the cell membranes of activated microglia, where it binds p47
phox subunit of NOX2-NADPH oxidase, in accordance with our previous studies in other phagocytic cells as well as in primary rat microglia [
40,
43,
48,
50]. The binding between p-cPLA
2α and 47
phox was detected at 15 min when the microglia cells were stimulated with LPS and at 4 h when stimulated with IFNγ in correlation with the detection of superoxide production and the kinetic of cPLA
2α phosphorylation by the two stimuli. Our previous study [
43] demonstrated that arachidonic acid activated the assembled oxidase in activated cPLA
2α-deficient cells, although the precise mechanism is not known. The restoration of CD40 upregulation in the activated cells that were pretreated with AS against cPLA
2α by addition of arachidonic acid is probably due to the activation of the NOX2-NADPH oxidase. The activation of cPLA
2α at 15 min by LPS and at 4 h by IFNγ was mediated by ERK activation since the presence of MEK inhibitor inhibited cPLA
2α activation in accordance with ours and other earlier studies [
44,
51].
The involvement of two transcription factors, NF-κB and STAT1α, was reported in the signal transduction pathways leading to induction of CD40 by either LPS or IFNγ [
37‐
39]. While NF-κB was shown to be rapidly activated by LPS, it was activated only at 4 h following exposure to IFNγ. In contrast, STAT1α was rapidly activated by IFNγ and only at 4 h by LPS. Time-dependent activation of cPLA
2α detected by its phosphorylation on serine 505 revealed that cPLA
2α is rapidly activated by LPS and only considerably later (4 h) by IFNγ, that is in accordance with a previous report [
44]. We show in the present study that the kinetic of activation of cPLA
2α coincided with the kinetic of NF-kB activation and that the activation of cPLA
2α is required for the activation of NF-κB in BV-2 microglia cell line, a finding consonant with our earlier study in microglia activated with amyloid beta [
40]. While superoxide production by NOX2-NADPH oxidase is extremely important for killing invading pathogens, it is also an important activator of diverse cell signaling pathways such as mitogen activated protein kinase and NF-κB to regulate the expression of genes encoding a variety of pro-inflammatory factors [
40,
52,
53]. The activation of NF-κB by either LPS or IFNγ shown in the present study detected by the phosphorylation of its p-65 subunit on serine 536 is probably mediated by superoxides produced by the NOX2-NADPH oxidase since the inhibition of the oxidase activity prevented NF-kB action. In line with this suggestion, the phosphorylation of p65 NF-kB RelA on Ser-536 is known to be redox-sensitive [
54]. The activation of NF-kB by NOX2-NADPH oxidase activity is consistent with our previous studies in microglia and macrophages [
40,
55] and with other in various systems and by various agonist [
56,
57].
It was reported that the activation of NF-kB under IFNγ stimulation is mediated by an autocrine effect of released TNF-α from the stimulated cells [
37]. Consistent with this observation, we show here that the activation of cPLA
2α and of NF-kB and the induction of CD40 by IFNγ are mediated by an autocrine effect of TNF-α, since TNF-α secretion from the activated cells was detected and the levels of secreted TNF-α activated cPLA
2α and NF-kB. In addition, the presence of antibodies against TNF-α in microglia stimulated with IFNγ of all three processes were inhibited, suggesting that cPLA
2α activation by TNF-α regulates the induction of CD40 via NF-kB activation. The activation of cPLA
2α by TNF-α coincided with other reports in microglia and macrophages [
46,
58]. However, addition TNF-α is not sufficient to induce CD40, although it activates cPLA
2α, probably since it stimulates the activation of NF-κB but not the activation of STAT1α that is also required for CD40 induction.
The activation of cPLA
2α and NF-κB in the signals leading to CD40 upregulation by LPS is mediated by both MyD88 and TRIF pathways, since inhibition of each pathway inhibited cPLA
2α and NF-κB activation and abolished CD40 induction. In accordance with our results, the activation of cPLA
2α by MyD88 and by TRIFF adaptive protein was shown in macrophages stimulated by LPS [
59]. The activation of NF-κB leading to CD40 upregulation by LPS was suggested to be mediated only by MyD88 adaptive protein in macrophages [
39]. However, several studies reported, similar to our results, that both pathways are mediating NF-κB by TLR4 receptor in macrophages and other cell types [
59‐
61].
Acknowledgements
We thank Dr. Sergio Lamprecht for assistance in editing the English text. This research was supported by a grant from the Israel Sciences Foundation founded by the Israel Academy of Sciences and Humanities 1012/09.