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The Journal of Immunology, 2007, 178: 720-731.
Copyright © 2007 by The American Association of Immunologists, Inc.

Guanosine Inhibits CD40 Receptor Expression and Function Induced by Cytokines and beta Amyloid in Mouse Microglia Cells1

Iolanda D’Alimonte*, Vincenzo Flati{dagger}, Mariagrazia D’Auro*, Elena Toniato{ddagger},§, Stefano Martinotti{ddagger},§, Michel P. Rathbone, Shucui Jiang||, Patrizia Ballerini*,§, Patrizia Di Iorio*,§, Francesco Caciagli*,§ and Renata Ciccarelli2,*,§

* Department of Biomedical Sciences, Section of Pharmacology, University of Chieti, Chieti, Italy; {dagger} Department of Experimental Medicine, University of L’Aquila, L’Aquila, Italy; {ddagger} Department of Oncology and Neuroscience, University of Chieti, Chieti, Italy; § Centre of Excellence on Aging, "G. D’Annunzio" Foundation, University of Chieti, Chieti, Italy; and Department of Medicine, Division of Neurology, and || Department of Surgery, McMaster University Health Sciences Centre, McMaster University, Hamilton, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Growing evidence implicates CD40, a member of the TNFR superfamily, as contributing to the pathogenesis of many neurodegenerative diseases. Thus, strategies to suppress its expression may be of benefit in those disorders. To this aim, we investigated the effect of guanosine, a purine nucleoside that exerts neurotrophic and neuroprotective effects. CD40 expression and function are increased by exposure of mouse microglia cultures or the N9 microglia cell line to IFN-{gamma} (10 ng/ml) plus TNF-{alpha} (50 ng/ml) or beta amyloid (Abeta) peptide (Abeta1–42; 500 nM). Culture pretreatment with guanosine (10–300 µM), starting 1 h before cytokine or Abeta addition, dose-dependently inhibited the CD40-induced expression as well as functional CD40 signaling by suppressing IL-6 production promoted by IFN-{gamma}/TNF-{alpha} challenge in the presence of CD40 cross-linking. Moreover, guanosine abrogated IFN-{gamma}-induced phosphorylation on Ser727 and translocation of STAT-1{alpha} to the nucleus as well as TNF-{alpha}-/Abeta-induced I{kappa}B{alpha} and NF-{kappa}B p65/RelA subunit phosphorylation, thus inhibiting NF-{kappa}B-induced nuclear translocation. Guanosine effects were mediated by an increased phosphorylation of Akt, a PI3K downstream effector, as well as of ERK1/2 and p38 in the MAPK system, because culture pretreatment with selective ERK1/2, p38 MAPK, and PI3K antagonists (U0126, SB203580, or LY294002, respectively) counteracted guanosine inhibition on IFN-{gamma}/TNF-{alpha}-induced CD40 expression and function as well as on STAT-1{alpha} or NF-{kappa}B nuclear translocation. These findings suggest a role for guanosine as a potential drug in the experimental therapy of neuroinflammatory/neurodegenerative diseases, particularly Alzheimer’s disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Microglia are the resident immunocompetent cells of the brain (1) involved in immune surveillance, host defense against microbial pathogens, and, more generally, in the reaction of the brain to injury (2). Under normal conditions, microglia display a quiescent phenotype, whereas, in response to brain injury, infection, or inflammation, microglia become highly activated, undergoing a process characterized by differentiation and proliferation. Activation of microglia is a histopathological hallmark of several neurodegenerative diseases, including Alzheimer’s and Parkinson’s diseases, AIDS dementia complex, multiple sclerosis, and amyotrophic lateral sclerosis (2, 3, 4, 5). Microglial activation is associated with an increase in the secretion of proinflammatory and neurotoxic mediators including cytokines, eicosanoids, oxygen radicals, and NO, which are believed to contribute to progressive damage in neurodegenerative disorders (reviewed by Refs. 1, 6 and 7).

CD40 is a phosphorylated 50-kDa glycoprotein that belongs to the TNF-{alpha} type I receptor superfamily (for review, see Ref. 8). It is expressed on the surface of various cells including monocytes and microglia (9, 10, 11, 12). The ligand for CD40 (named CD40L or CD154) is produced mainly by activated T lymphocytes, although other cells, such as vascular endothelial cells, smooth muscle cells, macrophages, and astrocytes have the capacity to synthesize and release CD40L. Several reports suggest that the interaction between CD40 and its cognate ligand CD40L, two costimulatory molecules critical for immune response regulation, plays a more general role in inflammatory processes (13). In particular, microglial CD40 ligation induces microglia maturation and the production of proinflammatory neurotoxic cytokines (14, 15, 16) and is therefore implicated in the initiation and/or progression of the neurodegenerative disorders mentioned above (17, 18, 19, 20).

It has been demonstrated that microglia constitutively express CD40 at a low level (21). IFN-{gamma}, which is a mediator of a number of proinflammatory effects and is one of the most potent microglia-activating factors, can up-regulate CD40 in different cell types, including mouse and human microglia in culture (1, 15). This induction is mediated by STAT-1{alpha} in cooperation with other transcription factors (22). More recently, it has been shown that a major component of IFN-{gamma}-induced CD40 expression involves the endogenous production of the cytokine TNF-{alpha} and the subsequent activation of the nuclear factor NF-{kappa}B (23). CD40 expression is also increased on cultured primary and N9 microglia treated with beta amyloid (Abeta)3 peptides and on microglia from a transgenic murine model (TgAPPSW) of Alzheimer’s disease (24). In addition, a low dose of IFN-{gamma} synergistically enhances Abeta-dependent CD40 expression on cultured microglial cells. Also in this case, the activated pathway could involve the stimulation of NF-{kappa}B, as it was reported that Abeta interaction with receptor for advanced glycation end products, expressed by activated microglia, led to NF-{kappa}B induction (25).

Because an aberrant expression of CD40 induced by cytokines or Abeta on microglia is directly correlated with pathogenic events occurring in neurodegenerative disorders (17, 24), strategies to suppress CD40 expression may attenuate inflammation and neuronal damage within the CNS, which will ultimately may be of benefit in neuroinflammatory diseases. IFN-{gamma}-induced CD40 expression in microglia is inhibited by pleiotrophins such as TGF-beta (21), cytokines such as IL-4 (26), or neuropeptides such as vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide (27). The inhibitory activity of these last factors has been related to an increase in the activity of the cAMP/protein kinase A transduction pathway. Other studies have pointed out the significant role played by the family of MAPKs, in particular ERK1/2 (28, 29) and p38 kinase (30, 31), in the control of the NF-{kappa}B-mediated signal, whereas the possible role of PI3K is still a matter of debate (32).

Guanosine is a purine nucleoside present extracellularly in brain both under physiological and pathological conditions (33, 34) in concentrations even higher than those of its better known adenine-based counterpart, adenosine. Guanosine exhibits anti-inflammatory and immunosuppressive properties (35, 36) and exerts a number of neurotrophic and neuroprotective effects, including astrocyte proliferation (37, 38), promotion of neurite outgrowth (39), increased synthesis, and release of neuro/pleiotrophins such as nerve growth factor, TGF-beta and basic fibroblast growth factor, from several cell types (40, 41), and enhanced glutamate uptake by astrocytes (42). Recently, we demonstrated that guanosine protects astrocytes against apoptotic death caused by staurosporine (43) and SH-SY5Y human neuroblastoma cells against apoptosis induced by Abeta peptides (44). The major part of guanosine-induced effects is linked to the activation of PI3K and MAPK pathways. In the present report, we investigated whether guanosine was able to suppress CD40 expression induced by various inflammatory stimuli in mouse microglia. We also attempted to identify the pathway(s) through which guanosine exerts these inhibitory effects.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Recombinant proteins, drugs, and reagents

Recombinant murine IFN-{gamma} and TNF-{alpha} were purchased from PeproTech. Guanosine, Abeta peptide (fragment1–42), pertussis toxin (PTX), 1,3- dipropyl-8-cyclopentylxanthine (DPCPX), 4-(2-[7-amino)]-2-(2-furyl(1,2,4)-triazolo(2,3-a (1,3,5)triazin-5-yl-aminoethyl)phenol) (ZM241385), suramin, and poly-D-lysine were supplied from Sigma-Aldrich, whereas [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride] (LY 294002) was from Tocris. [4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] (SB203580) and [1,4-diamino-2,3-dicyano-1,4-bis(2-quinophenylthio)butadiene] (U0126) were purchased from Calbiochem. Culture medium, antibiotics, and serum were obtained from Invitrogen Life Technologies. All other chemicals were of analytical grade or the best commercially available.

Microglia cultures

Microglia cultures were prepared according to the method recently published by Saura et al. (45). Briefly, mixed glial cultures were prepared from the cerebral cortex of 2- to 4-day-old CD11 mice (Charles River Laboratories), as previously reported (43). After enzymatic dissociation, cortical cells were seeded in DMEM with 10% FBS in poly-D-lysine-coated T75 flasks and cultured at 37°C in humidified 5% CO2/95% air. Medium was replaced every 3–4 days until confluence, which was achieved after 12–14 days in vitro. At that time, microglial cultures were prepared incubating mixed glial cultures with a trypsin solution (0.25% trypsin, 1.0 mM EDTA in HBSS) diluted 1/4 in DMEM for 10–20 min at 37°C. This treatment resulted in the detachment of astrocytes, leaving a population of firmly attached cells identified as >98% microglia, as previously described (45). Adherent microglia were grown in the usual culture medium for a further day and then submitted to evaluation. The N9 microglia cell line was a gift by Dr. P. Ricciardi-Castagnoli (Department of Biotechnology and Bioscience, University of Milano-Bicocca, Milan, Italy).

Western blot analyses

Western blot analysis was used to detect CD40, p65/RelA subunit, I{kappa}B{alpha}, and beta-actin as well as phosphorylated ERK1/2, I{kappa}B{alpha}, p65/RelA subunit (Ser536), p38 MAPK, Akt/protein kinase B (PKB), STAT-1{alpha} (Ser727), and STAT-1{alpha} (Tyr701).

Cultured microglia were serum-deprived for 24 h before pharmacological treatments (as reported in the figures). At the end of drug incubation, microglia were washed twice with ice-cold PBS and then harvested at 4°C in a lysis buffer (25 mM Tris buffer (pH 7.4), containing 150 mM NaCl, 100 µM sodium orthovanadate, 1.5 mM MgCl2, 1.0 mM EDTA, 1.0 mM EGTA, 1% Nonidet P-40, 10% glycerol, 1 mM PMSF, 5 µg/ml leupeptin, 5 µg/ml aprotinin). Cells were disrupted by sonication and then centrifuged at 14,000 rpm for 5 min at 4°C. Aliquots (20 µl) were removed from the supernatants for the determination of protein concentration by the method of Bradford (46). Samples were diluted in SDS-bromphenol blue buffer and boiled for 5 min. Cell lysates were separated on 12% SDS-PAGE and electrophoretically transferred to a polyvinylidene difluoride membrane (Bio-Rad). Membranes were incubated overnight at 4°C with specific primary Abs (polyclonal rabbit anti-phospho-ERK1/2, phospho-p38 MAPK, phospho-I{kappa}B{alpha}, phospho-p65 (Ser536), phospho-STAT-1{alpha} (Ser727), phospho-STAT-1{alpha} (Tyr701), phospho-Akt/PKB, from Cell Signaling, New England Biolabs, Celbio, all diluted 1/1000; polyclonal rabbit anti-p65, from Santa Cruz Biotechnology, diluted 1/100; and polyclonal rabbit anti-CD40 from Stressgen Biotechnologies, diluted 1/500). Membranes were then exposed to a secondary Ab for 1 h at room temperature (donkey anti-rabbit HRP-conjugated; Amersham Pharmacia Biotech). To confirm that equal amounts of protein were loaded in each lane, the membranes were incubated in stripping buffer (62.5 mM Tris-HCl (pH 6.7), 2% SDS, and 100 mM 2-ME) at 50°C for 30 min to remove the Abs. The blots were then reprobed with the nonphosphorylated form of the Abs mentioned above (dilution 1/1000, from Cell Signaling) or with rabbit polyclonal anti-beta actin (dilution 1/100; from Santa Cruz Biotechnology, incubation: 1 h at room temperature). Membranes were then exposed to a secondary Ab for 1 h at room temperature (donkey anti-rabbit HRP-conjugated from Amersham Pharmacia Biotech, diluted 1/2500), according to the manufacturer’s instructions. Immunocomplexes were visualized using the ECL detection system (Amersham Pharmacia Biotech). Densitometric analysis was performed for the quantification of the immunoblots, using the Molecular Analyst System (Bio-Rad) program.

Immunoprecipitation

Cell lysates were prepared as described for Western blot analysis with some modifications. Cells were harvested with buffer A (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF), centrifuged at 1200 rpm for 10 min and then resuspended with buffer B (20 mM HEPES, 0.4 M NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF). Immunoprecipitation was performed by incubating 0.5 mg of protein extracts with appropriate Ab (polyclonal rabbit anti-p65, from Santa Cruz Biotechnology, and polyclonal rabbit anti-I{kappa}B{alpha} from Cell Signaling, New England Biolabs) on ice for 30 min. Then, protein G-Sepharose (Amersham Pharmacia Biotech) was added and the samples rotated overnight at 4°C. Immunoprecipitated proteins were separated on a 7.5% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Bio-Rad). Immunostaining was performed using an appropriate secondary Ab for 1 h at room temperature (donkey anti-rabbit HRP-conjugated from Amersham Pharmacia Biotech, diluted 1/2500) and the immunoreactive protein bands were detected using ECL (Amersham Pharmacia Biotech).

EMSA

Microglia, after incubation with different drugs for various time periods, were harvested by scraping the cells off after washing with cold PBS, and pelleted to prepare nuclear extracts. EMSA was performed with 5–10 µg of nuclear extract in a total volume of 15 µl of binding buffer (50 mM NaCl, 1 mM MgCl2, 0.1 mM EDTA, 4% glycerol, 0.5 mM DTT, 4 mM Tris-HCl (pH 7.5), 1 µg of polydeoxyinosinic-deoxycytidyl acid, and 20,000 cpm [32P]oligonucleotide probe) and incubated on ice for 15 min. Bound and free DNA were then resolved by electrophoresis through a 6% polyacrylamide gel in 0.5x Tris-borate-EDTA buffer at 250 V for 1 h. The oligonucleotide sequence used as a probe included the distal NF-{kappa}B element (5'-CGAGGGAATTTCCTTTGAA) and the medial IFN-{gamma} activation sequence (GAS) element (5'-GGAAACTCTTCCTTGAAACGCCTCC) from the human CD40 promoter (23, 47). The underlined sequences are the canonical binding sites for the NF{kappa}B and GAS transcription factors.

Flow cytometry

After the treatment with cytokines for 24 h, in the presence or not of guanosine (pretreatment starting from 1 h before cytokine addition), microglial cells (5 x 105 cells) were harvested by trypsinization and washed twice with PBS. The cells were then incubated with 5 µl of 20 µg/ml conjugated mAb anti-mouse CD40-PE (R-PE) (Immunotech) at room temperature for 30 min in a dark room. Then, the cells were fixed and lysed with FACS lysing solution (BD Biosciences), washed with PBS, and analyzed in a BD Biosciences FACS flow cytometer using CellQuest software. The calibration was conducted with four colors using Calibrite3 plus allophycocyanin (BD Biosciences) and analyzed by FACScomp software.

RNA isolation and RT-PCR

Total RNA was isolated from confluent cells using TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer’s recommendations. The resulting RNA pellet was washed with 70% ice-cold ethanol, air dried, and redissolved in 30 µl diethyl-pyrocarbonate-treated water. The quantity and purity of RNA were estimated spectrophotometrically by absorbance at 260 nm, and 5 µg were run on formaldehyde gel to confirm the integrity of the RNA, as indicated by the preservation of the 28 and 18S rRNA.

To remove any genomic DNA contaminants, RNA samples (10 µg) were treated with 1 U of DNase-I RNase-free (Roche). First strand cDNA was synthesized from 1.5 µg of total RNA using the RT-PCR system RETROscript (Ambion) with random hexamers. The resultant cDNA (2 µg) was amplified in a 100-µl reaction volume containing PCR buffer, 1.5 mM MgCl2, 0.2 mM of each deoxy-dNTP, 1 µM oligonucleotide primers (MWG Biotech), 2.5 U of AmpliTaq Gold DNA polymerase (Applied Biosystems). The CD40 primer, 5'-CGGTGAAAGCGAATTCCTAG-3' (sense) and 5'-CAGCCTTCTTCACAGGTGC-3' (antisense) were synthesized by MWG Biotech. The 18S primer set was purchased from Ambion. Conditions applied for PCR amplification were 94°C for 5 min, 35 cycles of denaturation at 94°C for 1 min annealing at 55°C for 1 min, and elongation at 72°C for 1 min. Amplification products were resolved by 2% agarose gel electrophoresis. CD40 mRNA and 18S rRNA were detected by presence of 141- or 489-bp amplification products, respectively.

ELISA for IL-6 evaluation

Cultured microglial cells were plated in 24-well tissue culture plates at 1 x 105 cells/well and stimulated for 24 h with IFN-{gamma} (10 ng/ml) plus TNF-{alpha} (50 ng/ml) and an agonist anti-CD40 Ab (10 µg/ml; BD Biosciences/BD Pharmingen), in the presence or absence of guanosine. Cell-free supernatants were collected and stored at –80°C until analysis. IL-6 levels in the supernatants were examined by ELISA kit (R&D Systems) with detectable limits of 21–4000 pg/ml, in accordance to the manufacturer’s protocol. Cell lysates were also prepared and the Bio-Rad protein assay was performed to measure total cellular protein from each of the cell groups. Results are shown as mean picograms of cytokine per milligram of total cell protein.

Statistical analysis

All data are presented as the mean ± SEM for a series of n experiments. Statistical analyses were performed using the t test or ANOVA followed by the appropriate post hoc comparison. Group differences with p < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
In the present study, for most experiments, we used primary microglia cells derived from mouse brain or the N9 microglia cell line, in which IFN-{gamma}, in the presence of TNF-{alpha} or Abeta, has the ability to induce CD40 expression (23, 24). We examined the activity of guanosine on the effects of these proinflammatory stimuli. The concentrations of guanosine and its time of exposure did not affect the viability of those cells, as previously demonstrated for rat brain astrocytes (48).

Guanosine inhibits IFN-{gamma}/TNF-{alpha}- or IFN-{gamma}/Abeta-induced CD40 expression

To examine guanosine regulation on CD40 expression in microglial cells, cultured mouse microglia were treated with IFN-{gamma} (10 ng/ml) for different periods (6–48 h) and CD40 protein expression was measured by Western immunoblotting. Consistent with previous data of others (21, 22), IFN-{gamma} caused a robust time-dependent increase in the CD40 expression (Fig. 1A), that peaked at 24 h, remaining constant after 48 h. We also confirmed that IFN-{gamma}-induced CD40 expression, evaluated at 24 h, was remarkably augmented by either the presence of TNF-{alpha} (50 ng/ml) or Abeta (500 nM), as previously shown by Nguyen and Benveniste (23) or Tan et al. (24), respectively (Fig. 1B). Guanosine, added to the cultures at different concentrations, starting from 1 h before pharmacological treatments and maintained throughout the duration of the experiments, did not affect CD40 expression observed in basal condition. But guanosine did inhibit, in a concentration-dependent manner, the increases in expression of CD40 resulting from exposure of the cells to cytokines (Fig. 2A) or Abeta (data not shown). Additional experiments conducted on the N9 microglial cell line showed that guanosine caused an inhibitory effect on CD40-induced expression similar to that found in primary murine microglia. We confirmed the results obtained by immunoblot analysis using flow cytometry. Indeed, as shown in Fig. 2B, microglia exposure to cytokines for 24 h caused a remarkable increase in CD40 expression, as evaluated by a shift to the right in the fluorescence measurement (upper panel), whereas pretreatment with 300 µM guanosine abolished this effect (middle panel). We also confirmed that guanosine alone was unable to modify CD40 expression on cultured microglia (lower panel).


Figure 1
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  		FIGURE 1. CD40 expression induced by exposure of cultured mouse microglia to IFN-{gamma} alone or in combination with TNF-{alpha} or Abeta1–42 peptide. Mouse microglia, maintained for 24 h in serum-free culture medium, were exposed to IFN-{gamma} (10 ng/ml) for different times (A) or to IFN-{gamma} (10 ng/ml) and TNF-{alpha} (50 ng/ml) or Abeta1–42 peptide (500 nM) for 24 h (B). Subsequently, cell lysates were subjected to Western blotting using an Ab against CD40 and blots were reprobed with an Ab against beta-actin, to assure equal loading of samples. The results shown are representative of at least three experiments. Immunoblots were quantified by densitometric analysis and the CD40 values, normalized to the corresponding beta-actin values, are expressed as fold increase from basal value (untreated cells) in the histogram under the blots. Data are the means ± SEM of n = 3 independent experiments. *, p < 0.01; **, p < 0.001: statistical significance compared with the respective basal value.

 

Figure 2
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  		FIGURE 2. Culture pretreatment with guanosine (Guo) inhibits the expression and function of CD40 induced by microglia exposure for 24 h to the combined treatment with IFN-{gamma} and TNF-{alpha}. Mouse microglia, maintained for 24 h in serum-free culture medium, were pretreated with Guo at different concentrations, starting 1 h before IFN-{gamma} (10 ng/ml) and TNF-{alpha} (50 mg/ml) added alone (A and B) or in combination with an agonist anti-CD40 Ab (anti-CD40 mAb, 10 µg/ml) (C) for 24 h. Some cultures were exposed for the same period to Guo alone at the indicated concentrations. A, Cell lysates were immunoblotted with specific Abs against CD40 and reprobed with an Ab against beta-actin, to assure equal sample loading. Immunoblots, representative of three separate experiments, were quantified by densitometric analysis. The values of CD40, normalized to the corresponding beta-actin values, are expressed as fold increase from basal value in the histogram under the blots. B, At the end of microglia incubation with cytokines (24 h), in the presence or not of Guo pretreatment, cells were harvested and analyzed for the expression of CD40 by four-color flow cytometry. The filled area represents the background staining using an isotype-matched control Ab. The mean fluorescence intensities for cells in basal conditions (basal) or treated with cytokines or Guo alone (upper and lower panels) and in combination (middle panel) are located over an horizontal bar, indicating the positive staining, on the corresponding curves. The data shown are from one representative experiment of three total experiments. C, Microglia were seeded into 24 multiwell dishes and exposed for 24 h to the treatments described above. Supernatants were then collected to evaluate IL-6 release by an ELISA, following the manufacturer’s instructions. Data are expressed as picograms of cytokine per milligram of total cell protein. Values in histograms (A and C) are the means ± SEM of n = 3 separate experiments. *, p < 0.05; **, p < 0.005; ***, p < 0.001: statistical significance in comparison with basal value. §, p < 0.05; §§, p < 0.001: statistical significance as compared with value of IFN-{gamma}/TNF-{alpha} (±anti-CD40 mAb) treatment.

 
Guanosine inhibits functional CD40 signaling in microglia

To examine functional consequences of guanosine on CD40 expression, we stimulated mouse primary microglial cells with IFN-{gamma}/TNF-{alpha} plus CD40 cross-linking (agonist anti-CD40 Ab) (49, 50), in the presence or absence of guanosine, for 24 h. Supernatants from each treatment were examined by ELISA for IL-6 that has been described as being induced by microglial CD40 ligation (51). ELISA measurements revealed that guanosine per se was unable to cause IL-6 secretion over basal values, which were very low, probably due to the absence of a source of CD40L in the microglial cultures (19). In contrast, as we expected, IFN-{gamma}/TNF-{alpha}/CD40 cross-linking induced the secretion of large amounts of the proinflammatory molecule IL-6 (Fig. 2C). However, when microglia were stimulated in the presence of increasing concentrations of guanosine, the levels of this cytokine were reduced in a concentration-dependent fashion (Fig. 2C).

Effect of PTX or adenine-based purine receptor antagonists on the inhibitory activity of guanosine on IFN-{gamma}-/TNF-{alpha}-induced CD40 expression

Guanosine, a purine nucleoside, is not recognized as a ligand for adenosine P1 receptors. However, we have previously reported that guanosine increases the extracellular concentration of adenosine and adenine-based nucleotides, at least in cultured astrocytes (37, 41, 48). Because purines and adenosine, in particular, are considered endogenous immunomodulators, able to affect the signaling of inflammatory cytokines, including TNF-{alpha}, in different cell types (52), we wanted to ascertain whether the inhibitory effect of guanosine on cytokine-induced CD40 expression was due to adenosine or ATP. Therefore, we pretreated cultured microglia with antagonists for P1 and P2 purinoceptors, known to be present on microglia (53, 54). These antagonists, widely used to show selective effects caused by adenine-based purines on specific receptor sites (55, 56), have been used also by our group to demonstrate the involvement or not of these compounds in the effects promoted by guanosine (37, 43). In particular, we used DPCPX (100 nM) or ZM241385 (100 nM), selective antagonists of the A1 or A2A adenosine receptors, respectively, or suramin (10 µM), a nonselective antagonist of P2 ATP receptors, and administered them to the cultures starting 30 min before the addition of guanosine. DPCPX or suramin did not modify, whereas ZM241385 reduced, in part, the inhibitory effect of guanosine on IFN-{gamma}-/TNF-{alpha}-induced CD40 expression (Fig. 3). Additionally, microglial cultures were exposed to an overnight (16 h) pretreatment with PTX (200 ng/ml), a well-known inhibitor of metabotropic receptor-coupled Gi proteins. The inhibitory effect of guanosine on cytokine-induced expression of CD40 was reversed by PTX pretreatment, as shown by Western blot analysis (Fig. 3). These data provide evidence that the effect of guanosine on CD40 is in part mediated by activation of Gs protein-coupled A2A receptors by endogenous adenosine and in part appears to be linked to the stimulation of a Gi protein-coupled receptor.


Figure 3
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  		FIGURE 3. Effect of culture pretreatment with pertussis toxin (PTX) or P1 or P2 receptor antagonists on the inhibitory activity of guanosine (Guo) against the IFN-{gamma}/TNF-{alpha}-induced CD40 expression. Mouse microglia, maintained in serum-free medium for 24 h, were exposed for a further 24 h to IFN-{gamma} (10 ng/ml) and TNF-{alpha} (50 ng/ml), alone or in combination with Guo, administered 1 h before the two proinflammatory cytokines. The selective antagonists of adenosine A1 (DPCPX, 100 nM) and A2 (ZM241385, 100 nM) receptors or the nonselective antagonist of ATP P2 purinoceptor (suramin, 10 µM) were added to the cultures for 30 min whereas PTX (200 ng/ml) was added 16 h before Guo (300 µM). Cell lysates were then submitted to specific Western blot analysis and blots were reprobed with an Ab against beta-actin, to assure equal sample loading. The immunoblots, representative of three independent experiments with similar results, were quantified by densitometric analysis. The values of CD40, normalized to the corresponding beta-actin values, are expressed as fold increase from basal value in the histogram under the blots. Data are the means ± SEM of n = 3 experiments. *, p < 0.005: statistical significance as compared with basal value; §, p < 0.005; §§, p < 0.001 statistical significance in comparison with the effect of Guo.

 
Guanosine inhibits IFN-{gamma}-/TNF-{alpha}-induced CD40 expression on microglial cultures, acting at transcriptional level

To investigate whether the inhibitory effect of guanosine on CD40 expression promoted by the combined administration of IFN-{gamma} and TNF-{alpha} to microglia occurred at transcriptional level, we conducted a suitable RT-PCR experiment (Fig. 4). The assay demonstrated that mRNA transcription for CD40R was enhanced by microglial exposure to the two cytokines for different times and inhibited by cell pretreatment with guanosine. These data prompted us to extend our investigation to the nuclear level.


Figure 4
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  		FIGURE 4. Guanosine (Guo) inhibits microglial CD40 expression induced by IFN-{gamma}/TNF-{alpha}. Mouse microglial cells, serum starved for 24 h, were exposed to IFN-{gamma} (10 ng/ml) and TNF-{alpha} (50 ng/ml), in the presence or absence of 300 µM Guo, for different times. Total RNA was prepared from these cells and subjected to RT-PCR (see Materials and Methods) for analysis of CD40 mRNA expression. Mitochondrial RNA (18 S) was used as an internal control, to assure equal sample loading. M, m.w.; C, control (untreated cells).

 
Guanosine inhibits either IFN-{gamma}- or TNF-{alpha}- and Abeta-dependent STAT-1{alpha} or NF-{kappa}B nuclear translocation, respectively

We evaluated whether guanosine affected the nuclear signaling activated by IFN-{gamma} and TNF-{alpha} or Abeta in the promotion of CD40 expression. As assessed by EMSA assay on nuclear extracts from cells exposed to cytokines or Abeta peptide for 30 min (Fig. 5), guanosine pretreatment (starting from 1 h before cytokine or Abeta addition) markedly inhibited the ability of either IFN-{gamma}-activated STAT-1{alpha} to bind the oligonucleotide containing the GAS (Fig. 5A) or TNF-{alpha}-/Abeta-activated NF-{kappa}B to bind its sites (Fig. 5B), both present in the CD40 gene promoter. In contrast, guanosine by itself was unable to promote STAT-1{alpha} or NF-{kappa}B translocation into the nucleus. Very similar results were also obtained using the N9 microglia cell line (data not shown).


Figure 5
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  		FIGURE 5. Guanosine (Guo) opposes the nuclear translocation of the transcription factors STAT-1{alpha} and NK-{kappa}B induced by IFN-{gamma} and TNF-{alpha} or Abeta1–42 peptide, respectively. Microglia, serum-starved for 24 h, were exposed to IFN-{gamma} (10 ng/ml, A) or TNF-{alpha} (50 ng/ml) or Abeta1–42 peptide (500 nM, B) for 30 min. Where indicated, 300 µM Guo was added starting from 1 h before cytokine or Abeta peptide. Nuclear extracts were then prepared and subjected to EMSA, as indicated, to evaluate the nuclear translocation of the transcription factors STAT-1{alpha} (A) or of the nuclear factor NF-{kappa}B (B) induced by IFN-{gamma} or TNF-{alpha}/Abeta1–42 peptide on the corresponding binding sites (GAS and {kappa}B) present in the CD40 promoter gene. The results are representative of three independent experiments.

 
Guanosine inhibits IFN-{gamma}-dependent STAT-1{alpha} phosphorylation on serine 727

The translocation of STAT-1{alpha} to the nucleus is preceded by its phosphorylation on two residues, specifically Tyr701 or Ser727, which are both enhanced by microglia exposure to IFN-{gamma} (Fig. 6). Immunoblots conducted with specific Abs against each of the two residues showed that guanosine did not affect IFN-{gamma}-induced Tyr701 phosphorylation, but it markedly inhibited the phosphorylation of Ser727 promoted by IFN-{gamma}. In contrast, guanosine did not modify the activity of the suppressor of cytokine signaling 1, which is reported to inhibit IFN-{gamma}-induced activation of JAK1/2 and consequently STAT-1{alpha} (Tyr701) phosphorylation (data not shown).


Figure 6
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  		FIGURE 6. Effect of culture pretreatment with guanosine (Guo) on the time-dependent STAT-1{alpha} phosphorylation induced by IFN-{gamma} on Tyr701 and Ser727. Mouse microglia, serum starved for 24 h, were exposed to IFN-{gamma} (10 ng/ml) for different times, in the absence of Guo (positive control) or in the presence of 300 µM Guo, added 1 h before IFN-{gamma} treatment. Western blot analysis was conducted on cell lysates to evaluate STAT-1{alpha} phosphorylation on Tyr701 (A) and Ser727 (B). The immunoblots were reprobed with beta-actin, to assure sample equal loading, and quantified by densitometric analysis, the values of which, normalized to the corresponding values calculated for beta-actin, are reported as fold increase from basal value, in the histograms. Data are the means ± SEM of n = three separate experiments and immunoblots in the figure are representative of these experiments. *, p < 0.01; **, p < 0.005; ***, p < 0.001: statistical significance as compared with the respective basal value (untreated cells). §, p < 0.01; §§, p < 0.005: statistical significance in comparison with positive control.

 
Guanosine inhibits TNF-{alpha}- and Abeta-dependent I{kappa}B{alpha} phosphorylation and NF-{kappa}B subunit p65 (RelA) activation

The formation of the active NF-{kappa}B complex, constituted by p65/RelA and p50 subunits, and its subsequent translocation to the nucleus is preceded by the phosphorylation of I{kappa}B{alpha}, a protein that normally sequesters NF-{kappa}B complex in the cytosol in an inactive form. Once phosphorylated, I{kappa}B{alpha} undergoes ubiquitination and proteolytic degradation (57). To determine whether guanosine had any effect on the TNF-{alpha}- or Abeta-induced I{kappa}B{alpha} phosphorylation, we examined the levels of the Ser32 hyperphosphorylated form of I{kappa}B{alpha}. Western blot analysis using a phosphospecific I{kappa}B{alpha} Ab revealed that TNF-{alpha} or Abeta induced I{kappa}B{alpha} phosphorylation as early as 5 min (Fig. 7A). At the same time, these treatments caused a corresponding decrease in the nonphosphorylated I{kappa}B{alpha} levels, as evaluated by immunoprecipitation with a specific Ab for p65/RelA subunit and subsequent immunoblotting using an anti-I{kappa}B{alpha} Ab (Fig. 7B). Guanosine counteracted both these effects caused by exposing microglia to TNF-{alpha} or Abeta (Fig. 7, A and B). Additionally, we analyzed the TNF-{alpha}- or Abeta-induced phosphorylation of p65/RelA subunit at Ser536, which is required for efficient transcriptional activation of NF-{kappa}B. Consistent with results reported above, guanosine pretreatment suppressed the phosphorylation of p65/RelA induced by TNF-{alpha} or Abeta after 5 min of treatment (Fig. 7C). Altogether, these results indicate that guanosine inhibited TNF-{alpha} or Abeta-induced NF-{kappa}B-dependent transcription activity, by blocking the signal transduction pathway involved in the activation of NF-{kappa}B nuclear translocation.


Figure 7
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  		FIGURE 7. Effect of guanosine (Guo) on the phosphorylation of I{kappa}B{alpha} and NF-{kappa}B complex p65 subunit and on the changes in the content of I{kappa}B{alpha} caused by microglia exposure to TNF-{alpha} or Abeta1–42 peptide. Mouse microglia, serum-starved for 24 h, were exposed to TNF-{alpha} (50 ng/ml) or Abeta1–42 peptide (500 nM) for 5 and 15 min (positive control). Guo (300 µM) was added starting from 1 h before these treatments. Cell lysates were subjected to Western blotting using Abs against phospho-I{kappa}B{alpha} (Ser32) (A) and phospho-p65 (RelA) subunit of the activated complex NF-{kappa}B (C). In parallel, aliquots of cells lysates were immunoprecipitated by an anti-p65 Ab and then analyzed by Western blotting using an Ab against the nonphosphorylated I{kappa}B{alpha} (B). The blots were then reprobed with Abs against beta-actin or p65 subunit, to assure equal sample loading, and quantified by densitometric analysis, the values of which are reported in the histograms as fold increase from basal value. Data are the mean ± SEM of three independent experiments with similar results. *, p < 0.05; **, p < 0.005; ***, p < 0.001: statistical significance as compared with the corresponding basal value (untreated cells). §, p < 0.05; §§, p < 0.01; §§§, p < 0.001: statistical significance as compared with the respective positive control.

 
Guanosine activates p38 and ERK1/2 MAPK as well as PI3K kinase pathways

To elucidate the signaling activated by guanosine and involved in its effects on cytokine-induced CD40 expression and function, we examined the activity of guanosine, in comparison with that of IFN-{gamma} and TNF-{alpha}, on different molecular pathways. Immunoblots reported in Fig. 8A show that guanosine, like IFN-{gamma} or TNF-{alpha}, increased p38 phosphorylation, which correlates well with stimulation of kinase activity (58, 59). However, the comparison among the time courses of p38 activation by IFN-{gamma} or TNF-{alpha} and guanosine shows that induction of p38 activity by guanosine was more intense and more sustained than that caused by the other two agents.


Figure 8
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  		FIGURE 8. Time courses of the phosphorylation of p38 and ERK1/2 MAPK and Akt induced by IFN-{gamma}, TNF-{alpha}, and guanosine (Guo). Mouse microglia, after serum-starvation for 24 h, were exposed to IFN-{gamma} (10 ng/ml), TNF-{alpha} (50 ng/ml), or Guo (300 µM) for different times. Cell lysates were subjected to specific Western blotting using Abs against phospho-p38, phospho-ERK1/2, and phospho-Akt (A–C). Immunoblots, reprobed with Abs against beta-actin or nonphosphorylated ERK1/2 and Akt, to assure equal sample loading, were quantified by densitometric analysis, the values of which, normalized to beta-actin or nonphosphorylated ERK1/2 and Akt values, are reported in the histograms. Data are the mean ± SEM of three separate experiments. *, p < 0.05; **, p < 0.005; ***, p < 0.0001: statistical significance in comparison with basal value (untreated cells).

 
Likewise, guanosine and IFN-{gamma} or TNF-{alpha} stimulated ERK1/2 phosphorylation (Fig. 8B). Also in this case, the temporal profile of ERK1/2 activation induced by guanosine or cytokine stimulation was different. In fact, guanosine caused a very rapid and sustained increase in ERK1/2 activation, with a second peak at 60 min, whereas IFN-{gamma} and TNF-{alpha} caused a delayed stimulation of ERK1/2 at 30 min (Fig. 8B).

Finally, because we previously found in cultured rat astrocytes that guanosine activated the PI3K pathway, we investigated whether this was also true in mouse microglia. Indeed guanosine, rapidly increased phosphorylation of Akt, the best known downstream effector of PI3K (Fig. 8C). Neither IFN-{gamma} nor TNF-{alpha} was able to induce Akt phosphorylation. As expected, when guanosine was added to the cultures 1 h before the single addition of IFN-{gamma} or TNF-{alpha}, the observed effects were similar to those elicited by guanosine alone on p38, ERK1/2 (Fig. 9) and Akt (data not shown), because the increases in the phosphorylation of these kinases occurred earlier and were more sustained that that caused by the two cytokines.


Figure 9
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  		FIGURE 9. Time courses of the phosphorylation of p38 and ERK1/2 MAPK induced by IFN-{gamma} or TNF-{alpha} in combination with guanosine (Guo). Mouse microglia, after serum starvation for 24 h, were exposed to IFN-{gamma} (10 ng/ml) or TNF-{alpha} (50 ng/ml) in combination with Guo (300 µM) for different times. Cell lysates were subjected to specific Western blotting using Abs against phospho-p38 and phospho-ERK1/2 (A and B). Immunoblots, reprobed with Abs against beta-actin, to assure equal sample loading, were quantified by densitometric analysis, the values of which, normalized to beta-actin, are reported in the histograms. Data are the means ± SEM of three separate experiments. *, p < 0.05; **, p < 0.005; ***, p < 0.0001: statistical significance in comparison with basal value (untreated cells).

 
Activation of p38 and ERK1/2 MAPK and PI3K/Akt pathways by guanosine has a role in the inhibition of IFN-{gamma}-/TNF-{alpha}-stimulated CD40 expression and function

We investigated whether the molecular pathways activated by guanosine were involved in the inhibitory effect shown by the nucleoside on cytokine-induced CD40 expression and function. We pretreated microglial cultures with specific inhibitors of p38 and ERK1/2 MAPK or of PI3K pathways (SB203580, U0126, or LY294002, respectively), before guanosine addition. SB203580 and U0126 significantly reduced whereas LY294002 nullified the inhibitory effect of guanosine on either cytokine-stimulated CD40 expression or IL-6 secretion induced by IFN-{gamma}/TNF-{alpha}/CD40 cross-linking (Fig. 10, A and B). Accordingly, the inhibitory effects of guanosine on IFN-{gamma}-/TNF-{alpha}-induced STAT-1{alpha} and NF-{kappa}B nuclear translocation were substantially reduced by culture pretreatment with LY294002, and to a lesser extent, by SB203580 or U0126 (Fig. 10C). Interestingly, SB203580, but not LY294002 or U0126, significantly reduced I{kappa}B{alpha} phosphorylation caused by guanosine, indicating a prevailing activity of p38 kinase on this step in the NF-{kappa}B-signaling cascade (Fig. 10D). Altogether, these results indicate that guanosine controls IFN-{gamma}-/TNF-{alpha}-induced CD40 expression and function as well as STAT-1{alpha} and NF-{kappa}B activation via the stimulation of PI3K, p38, and ERK1/2 pathways.


Figure 10
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  		FIGURE 10. Effect of inhibitors of p38 MAPK, MEK1/2, and PI3K on the inhibitory activity of guanosine (Guo) on the expression and function of CD40 as well as on the nuclear translocation of transcription factors induced by microglia exposure to IFN-{gamma}/TNF-{alpha}. Mouse microglia, after serum-starvation for 24 h, were exposed to different treatments. A, IFN-{gamma} (10 ng/ml) and TNF-{alpha} (50 ng/ml) were added for further 24 h (positive control). Guo (300 µM) pretreatment started 1 h before this treatment and the inhibitors of p38 MAPK (SB203580 10 µM), MEK1/2 (U0126, 10 µM), and PI3K (LY294002, 30 µM) were added 30 min before Guo. Cell lysates were then subjected to Western blotting to evaluate CD40 expression and blots were reprobed with an Ab against beta-actin, to assure equal sample loading. B, Microglia were seeded into 24-well culture dishes and exposed to the combined treatment with IFN-{gamma}, TNF-{alpha}, and an agonist Ab anti-CD40 (10 µg/ml, anti-CD40 mAb) (positive control). Guo and kinase inhibitors mentioned above were added before IFN-{gamma} and TNF-{alpha}, as described for A. Supernatants were collected to evaluate IL-6 release by ELISA. Data are expressed as picograms of cytokine per milligram of total cell protein. C and D, IFN-{gamma} or TNF-{alpha} were administered to the cultures for 30 (C) or 15 (D) min, whereas Guo and kinase inhibitors were added before cytokines, as described above. Nuclear extracts were subjected to EMSA to evaluate the nuclear translocation of the transcription factors STAT-1{alpha} and NF-{kappa}B, whereas cell lysates were subjected to Western blotting, using Abs against phospho-I{kappa}B{alpha} (Ser32). The blots were then reprobed with Abs against beta-actin to assure equal sample loading. The immunoblots of A and D were quantified by densitometric analysis, the values of which are reported in the histograms as fold increase from basal value. Values in the histograms of A, B, and D are the means ± SEM of three independent experiments. *, p < 0.001: statistical significance as compared with basal value; #, p < 0.01; ##, p < 0.0005: statistical significance in comparison with the respective positive control; §, p < 0.05; §§, p < 0.01: statistical significance as compared with the effect of Guo alone.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
CD40 expression is pivotal for disease initiation and progression in experimental allergic encephalomyelitis, a mouse model of multiple sclerosis (58, 59, 60). In addition, aberrant expression of CD40 in microglia has been directly correlated with pathogenic events occurring in the CNS of patients with multiple sclerosis (61, 62, 63). In Alzheimer’s disease, activated microglia are considered a major contributor to the local inflammatory responses observed in neuritic plaques. Several studies have reported an increase in CD40-positive microglia as well as an enhancement of CD40L expression on astrocytes in Alzheimer’s disease (19, 64). Additionally, it has been shown that an Abeta challenge induces microglial CD40 expression, the ligation of which results in the production of neurotoxins (24). An in vivo validation of a pathogenic role for microglial CD40 was shown by the beneficial effects of the blockade of CD40 signaling in AD mouse models (65). Given the growing evidence of the importance of CD40 and its ligation by CD40L in neurological diseases, we have been particularly interested in the regulation of CD40 expression and function. The findings of the present study demonstrate that guanosine reduces IFN-{gamma}-/TNF-{alpha}- or Abeta-induced CD40 expression and function in mouse microglial cells and the relevance of these data should be considered in the context of the function of microglial CD40 in conditions described above.

In the present study, the effect of guanosine was mediated in part by adenosine. As we previously demonstrated (37, 41, 48), guanosine promotes a prompt release of great amounts of adenine-based nucleotides from glial cells, which are rapidly converted into adenosine and other metabolites by specific ectoenzymes located on the cell membrane surface (40). Based on this evidence, we always verified whether adenine-based purines were involved in the effects promoted by guanosine. Thus, we found that adenosine is not implicated in the guanosine-induced antiapoptotic effect in astrocytes (43) or neurite outgrowth in PC12 cells (39), whereas it contributes to the increase in cell proliferation (37) or cAMP intracellular levels promoted by guanosine in astrocytes (66). In both cases, the adenosine activity was mediated via A2Rs. Interestingly, adenosine, acting on the same receptor subtype, plays a significant role as immunomodulatory agent (36), also inhibiting the TNF-induced NF-{kappa}B activation in cell types other than microglia. However, the mechanism underlying this effect is not linked to the inhibition of TNF-{alpha}-induced I{kappa}B{alpha} phosphorylation/degradation, as we reported for guanosine here (52). Because PTX prevents the inhibition by guanosine on cytokine-induced CD40 expression, we speculate that guanosine exerts a part of its activity by interacting with Gi protein-linked receptors, which might correspond to the specific binding sites, the existence of which we demonstrated in membranes deriving from rat whole brain and astrocytes (41, 67). Of course, specific receptor cloning or availability of selective antagonists for guanosine will allow to demonstrate this aspect. At present, we cannot rule out that other substances, secreted by microglia under guanosine challenge, may interfere with Gi protein-linked receptors, thus mediating the modulatory effects of guanosine on CD40.

Interestingly, the effect of guanosine in reducing CD40 expression is linked to its ability to abolish either the activation of IFN-{gamma}-dependent STAT-1{alpha} or TNF-{alpha}- and Abeta-dependent NF-{kappa}B activation. In particular, the effect on STAT-1{alpha} activation was achieved by reducing the phosphorylation on serine 727, but not on tyrosine 701. Indeed, Tyr701 phosphorylation is essential for the nuclear translocation and DNA binding of STAT-1{alpha}, but the full transcriptional activity of STAT-1{alpha} is manifested when the Ser727 site is phosphorylated (68, 69). Additionally, guanosine inhibits the activation of NF-{kappa}B in microglia resulting from challenge by TNF-{alpha} or Abeta. The inhibition occurs not only on the NF-{kappa}B binding to DNA, but also on the upstream pathway leading to NF-{kappa}B activation. Some other agents have been demonstrated to inhibit the activation of STAT-1 or NF-{kappa}B induced by IFN-{gamma} or TNF-{alpha}, respectively (27, 51, 70, 71), but this is the first time that a single agent has been shown to be capable of inhibiting both pathways.

Another interesting aspect is that guanosine causes its inhibitory effects by activating multiple signaling pathways. We showed that guanosine activated both the p38 and ERK1/2 MAPK as well as the PI3K system by acting on STAT-1{alpha} and NF-{kappa}B signaling to inhibit CD40 expression and function. In fact, SB203580, U0126, and LY294002, specific inhibitors of these pathways, were able to counteract guanosine effects, acting at different levels. In particular, our data indicate that the influence of p38 kinase activation by guanosine on TNF-{alpha}-induced NF-{kappa}B signaling is prevalent on the phosphorylation of I{kappa}B{alpha}, whereas that of PI3K or ERK1/2 MAPK is downstream, on the translocation of the TNF-{alpha}-activated NF-{kappa}B complex into the nucleus. Our data are in agreement with findings showing that persistent activation of p38 may be associated with inhibition of IFN-{gamma} or TNF-{alpha} signaling (30, 31, 72). As well, it has been demonstrated that the ERK1/2 MAPK pathway plays a negative role on NF-{kappa}B, at least when activation is induced by inflammatory stimuli such as IL-1, TNF-{alpha}, or LPS (28, 29). In contrast, p38 and ERK1/2 MAPK were in part activated also by IFN-{gamma} or TNF-{alpha}. This has also been demonstrated by others in various cell models (73, 74, 75, 76, 77, 78). These seemingly incongruous effects may be attributable to the kinetics and extent of activation of the MAPK system by guanosine, which overcame the stimulation caused by the two cytokines, when administered to the cultures in combination with IFN-{gamma} or TNF-{alpha} (Fig. 9). In addition, it is important to emphasize the role played by the PI3K system, which, in our study, was activated only by guanosine. The PI3K/Akt/PKB pathway was first implicated in cell survival, when Yao and Cooper (79) reported that inhibitors of PI3K blocked nerve growth factor-dependent survival of PC12 cells. Since that time, the importance of this pathway in cell growth and survival has been widely documented (80, 81, 82, 83, 84, 85). We have recently shown that guanosine counteracts staurosporine- or Abeta-induced apoptosis in rat astrocytes or human neuroblastoma cells, respectively (43, 44) by activation of PI3K cascade. This pathway also inhibits apoptosis triggered by TNF-{alpha} in endothelial cells (86). However, the possible role of PI3K signaling in cytoplasmic NF-{kappa}B activation is still a matter of debate (32). Indeed, a number of research groups found no evidence for the involvement of the PI3K system in NF-{kappa}B-mediated gene expression in endothelial cells, smooth muscle cells, or fibroblasts (87, 88, 89). Thus, to our knowledge, this is the first report that identifies this enzyme cascade in the control of transcription factor phosphorylation at the level of gene promoters consequent to cytokine or Abeta stimulation.

In conclusion, we showed that guanosine decreases CD40 expression and function in cultured mouse microglial cells. By reducing CD40 expression, guanosine may reduce inflammation, a feature believed to be important in many neurodegenerative disorders. In particular, the present data, along with the evidence that guanosine in vitro is also able to counteract apoptosis caused by Abeta in neuronal cells (44), raise the possibility that guanosine could be used as an agent for the treatment of Alzheimer’s disease.


   Acknowledgments
 
We thank Dr. P. Ricciardi Castagnoli (University of Milan, Milan, Italy) for her gift of the N9 microglia cell line; Dr. Vincenzo Dadorante (Department of Oncology and Neuroscience, University of Chieti, Chieti, Italy) and Dr. Silvana Buccella (Department of Biomedical Sciences, University of Chieti, Chieti, Italy), for help with flow cytometry analysis and RT-PCR, respectively.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by funds to R.C. and F.C. from the Centre of Excellence on Aging of the University of Chieti and from the Italian Ministry of Education, University and Research. Back

2 Address correspondence and reprint requests to Dr. Renata Ciccarelli, Section of Pharmacology, Department of Biomedical Sciences, Medical School, University of Chieti, Via dei Vestini 29, pal. B. 66013 Chieti, Italy. E-mail address: r.ciccarelli{at}dsb.unich.it Back

3 Abbreviations used in this paper: Abeta, beta amyloid; PTX, pertussis toxin; DPCPX, 1,3-dipropyl-8-cyclopentylxanthine; ZM241385, 4-(2-[7-amino)]-2-(2-furyl(1,2,4)- triazolo(2,3-a(1,3,5)triazin-5-yl-amino ethyl)phenol); LY294002, [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydro chloride]; SB203580, [4-(4-fluorophenyl)-2-(4-methylsulfinyl phenyl)-5-(4-pyridyl)1H-imidazole]; U0126, [1,4-diamino-2, 3-dicyano-1,4-bis(2-quino phenylthio) butadiene]; PKB, protein kinase B; GAS, IFN-{gamma} activation sequence. Back

Received for publication November 30, 2005. Accepted for publication October 27, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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