Adenosine A2B receptor-mediated leukemia inhibitory factor release from astrocytes protects cortical neurons against excitotoxicity
© Moidunny et al.; licensee BioMed Central Ltd. 2012
Received: 19 March 2012
Accepted: 1 August 2012
Published: 16 August 2012
Neuroprotective and neurotrophic properties of leukemia inhibitory factor (LIF) have been widely reported. In the central nervous system (CNS), astrocytes are the major source for LIF, expression of which is enhanced following disturbances leading to neuronal damage. How astrocytic LIF expression is regulated, however, has remained an unanswered question. Since neuronal stress is associated with production of extracellular adenosine, we investigated whether LIF expression in astrocytes was mediated through adenosine receptor signaling.
Mouse cortical neuronal and astrocyte cultures from wild-type and adenosine A2B receptor knock-out animals, as well as adenosine receptor agonists/antagonists and various enzymatic inhibitors, were used to study LIF expression and release in astrocytes. When needed, a one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test was used for statistical analysis.
We show here that glutamate-stressed cortical neurons induce LIF expression through activation of adenosine A2B receptor subtype in cultured astrocytes and require signaling of protein kinase C (PKC), mitogen-activated protein kinases (MAPKs: p38 and ERK1/2), and the nuclear transcription factor (NF)-κB. Moreover, LIF concentration in the supernatant in response to 5′-N-ethylcarboxamide (NECA) stimulation was directly correlated to de novo protein synthesis, suggesting that LIF release did not occur through a regulated release pathway. Immunocytochemistry experiments show that LIF-containing vesicles co-localize with clathrin and Rab11, but not with pHogrin, Chromogranin (Cg)A and CgB, suggesting that LIF might be secreted through recycling endosomes. We further show that pre-treatment with supernatants from NECA-treated astrocytes increased survival of cultured cortical neurons against glutamate, which was absent when the supernatants were pre-treated with an anti-LIF neutralizing antibody.
Adenosine from glutamate-stressed neurons induces rapid LIF release in astrocytes. This rapid release of LIF promotes the survival of cortical neurons against excitotoxicity.
Keywords5′-N-Ethylcarboxamide (NECA) Leukemia inhibitory factor Neuroprotection Glutamate
Leukemia inhibitory factor (LIF) is a soluble glycoprotein that belongs to the family of interleukin (IL)-6-type cytokines. Other members of this family include IL-6, IL-11, ciliary neurotrophic factor (CNTF), oncostatin M (OSM), cardiotrophin-1 (CT-1) and novel neurotrophin-1 (NNT-1) , which display pronounced trophic as well as protective properties during pathophysiology of the central nervous system (CNS) and are hence referred to as neuropoietic cytokines or neurokines . Specific functions of LIF in the nervous system include induction of cholinergic differentiation of sympathetic neurons, induction of neuropeptide and choline acetyltransferase (ChAT) gene expression , regulation of polyneuronal innervation of neuromuscular junction [4, 5] and regulation of the HPA axis [6, 7]. Furthermore, LIF signaling is crucial for development of the nervous system, including development of sensory and motor neurons [8, 9] and glial cells . Consistently, reduced numbers of astrocytes and oligodendrocytes are found in LIF knock-out mice . During inflammation, LIF has been suggested to be both pro- and anti-inflammatory and appears to play a key role in neural injury and regeneration. We and others have previously demonstrated the neuroprotective properties of LIF against damages caused by excitotoxicity, light, et cetera[12–14]. Moreover, promotion of axonal regeneration and oligodendrocyte growth and survival by LIF suggests its potential for reducing damage associated with central inflammatory demyelinating diseases such as multiple sclerosis [15–17].
In the CNS, astrocytes are considered to be the major source for LIF [18, 19], and its expression in the brain is significant during pathological conditions including ischemia [20, 21], multiple sclerosis , Alzheimer’s and Parkinson’s diseases  and brain injury . The factors responsible for elevated LIF induction during CNS pathology are largely unknown. One of the candidates identified recently to induce LIF expression in astrocytes is ATP [18, 25], levels of which also rise during conditions like high-frequency neuronal activity, seizure, ischemia and hypoxia [26, 27]. However, extracellular ATP is rapidly hydrolyzed by a cascade of ectonucleotidases resulting in an enhanced level of adenosine [26, 28]. Correspondingly, excitotoxic conditions such as ischemia, hypoxia, seizure and head injury are known to induce a rapid increase in extracellular adenosine concentrations, up to 100 times that of the resting concentration [29–33]. There is abundant evidence for immune regulation by adenosine  including expression and release of growth factors and cytokines such as nerve growth factor (NGF), S100beta, IL-6 and CCL2 in glial cells [35–39]. However, it is not known whether adenosine can induce LIF expression in astrocytes.
In the present study, we investigated the potential influence of adenosine receptor activity on LIF release from cultured astrocytes.
Chemicals and reagents
Neurobasal media, Hank’s balanced salt solution (HBSS), phosphate-buffered saline (PBS), sodium pyruvate, L-glutamine, penicillin-streptomycin, hydroxyethyl piperazineethanesulfonic acid (HEPES), glutaMAX-1 and B27 supplement were obtained from Gibco (Breda, The Netherlands). Dulbecco’s modified Eagle’s medium (DMEM) and fetal calf serum (FCS) were obtained from PAA Laboratories (Cölbe, Germany). Trypsin was obtained from Life Technologies (Breda, The Netherlands). L-leucine methyl ester (LME) and the remaining cell medium components were purchased from Sigma-Aldrich (Zwijndrecht, The Netherlands). Recombinant mouse LIF (rmLIF: LIF2005) was obtained from Millipore (Amsterdam, The Netherlands). Brefeldin A (BFA), caffeine, L-glutamate, adenosine A2B receptor antagonist (MRS 1754), protein kinase A (PKA) inhibitor (KT 5720), protein kinase C (PKC) inhibitor (Ro 31–8220), p38 mitogen-activated protein kinases (MAPK) inhibitor (SB 203580), and adenosine analog (5′-N-Ethylcarboxamide or NECA) were obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands). Non-hydrolysable ATP (2MeSATP), adenosine A2A receptor antagonist (ZM 241385), adenosine A2A receptor agonist (CGS 21680) and MEK1/2 inhibitor (U 0126) were obtained from Tocris Bioscience (Bristol, UK). NF-kB inhibitor (BAY 11–7082) and c-Jun N-terminal kinase (JNK) inhibitor (SP 600125) were obtained from Calbiochem (Darmstadt, Germany). Reagents used in immunoblotting experiments were purchased from Bio-Rad Laboratories (Veenendaal, The Netherlands) with the exception of the polyvinylidene fluoride (PVDF) membranes that were obtained from Millipore (Bedford, MA).
Wild-type C57BL/6 J (1 to 2 days postnatal) mice were obtained from Central Laboratory Animal Facility (University of Groningen, The Netherlands). Adenosine A2B receptor knock-out (A2B KO) mice (1 to 2 days postnatal) with the same genetic background were kindly provided by Professor Marco Idzko (University of Freiburg, Germany). Wild-type C57BL/6 J (14 to 15 days embryonic) mice were obtained from Harlan (Horst, The Netherlands). All procedures were in accordance with the regulation of the Ethical Committee for the use of experimental animals of the University of Groningen, The Netherlands (License number DEC 4623A and DEC 5913A). Animals were housed in standard Makrolon™TM (Bayer AG, Leverkusen, Germany) cages and maintained on a 12 hour light/dark cycle. They received food and water ad libitum.
Primary neuronal culture
Primary culture of cortical neurons from mouse embryo (~E15) was established as described previously . Briefly, cortices from embryonic brains were dissected in ice-cold HBSS supplemented with 30% glucose. Meninges were removed, and the tissues were treated with trypsin before they were gently dissociated by trituration in neuronal culture media (neurobasal medium supplemented with 2% B27, 1 mM sodium pyruvate, 2 mM L-glutamine and 50 U/mL penicillin-streptomycin). The cell suspension was filtered using cell strainer (70 μm) (BD Falcon, Franklin Lakes, NJ, USA) before centrifugation (800 rpm for 10 minutes). Cells were then seeded on poly-D-lysine-coated six-well plates (1.5 x 106 cells/well) and maintained in neuronal culture media in a humidified atmosphere with 5% CO2 at 37°C. The culture medium was refreshed the next day to get rid of debris. The neuronal purity as determined by Microtubule-associated protein 2 (MAP2)-staining was around 98% (data not shown) . Cultures were used after 5 days in vitro.
Induction of excitotoxicity
Cortical neuron cultures were subjected to an excitotoxic challenge with glutamate (50 μM, for 1 hour), after which cultures were refreshed with fresh media and were incubated at 37°C. Supernatants from neuron cultures (untreated and glutamate-treated) were collected 18 hours after glutamate challenge and were applied to the primary astrocyte cultures.
Primary astrocyte cultures
Primary astrocyte cultures were established from cerebral cortices of postnatal (1 to 2 days) C57BL/6 J and A2B KO mice according to a previously described procedure , which was modified to reduce microglial contamination . Microglial cells were separated from the astrocytic monolayer by 1-hour shake-off at 150 rpm. This procedure was repeated two times with an interval of 4 days in vitro between each shake off, followed by an overnight shake-off at 240 rpm to remove oligodendrocyte precursor cells. Purified astrocytes were washed with HBSS buffer containing 1 mM ethylenediaminetetraacetic acid (EDTA) and further detached using HBSS with 0.1% trypsin. Cells were reseeded with fresh astrocyte culture medium (DMEM supplemented with 5% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate and 50 U/mL penicillin-streptomycin) in multi-well plates (5 x 104 cells/cm2) and maintained in culture to confluency. To further reduce microglial contamination, confluent astrocyte cultures were treated with 5 mM LME, a lysosomotropic agent , for 4 to 5 hours. Astrocytes were ready for experiments after 1 to 2 days. Our cell preparations had a high percentage of astrocytes (≥95%), which was confirmed by immunostaining against GFAP (astrocyte specific marker) and CD11b (microglial specific marker) (data not shown).
Real-time polymerase chain reaction
and was used to determine the relative gene expression levels .
Western blotting on cultured cortical astrocytes was performed as previously described . Equal amounts of protein (30 μg) were loaded to 12.5 or 15% sodium dodecyl sulfate-polyacrylamide gels and subsequently transferred to PVDF membranes. The membranes were blocked using Odyssey™ Blocking Buffer (OBB; LI-COR Biosciences, Cambridge, UK; diluted 1:1 in PBS) for 1 hour and incubated overnight at 4°C with different combinations of primary antibodies (diluted in 1:1 OBB and PBS + 0.1% Tween 20 (PBS-T)): mouse monoclonal anti-β-actin (1:8000, Abcam, Cambridge, UK); rabbit monoclonal anti-phospho-NF-κB p65 (Ser536) (1:1000, Cell Signaling Technology, Leiden, The Netherlands); and rat monoclonal anti-LIF (MAB449; 1 μg/mL, R&D Systems, Oxford, UK). The next day, membranes were washed in PBS-T (four times for 5 minutes each time) and incubated for 1 hour at room temperature with appropriate fluorescence conjugated secondary antibodies (diluted in PBS-T): donkey anti-mouse IR Dye 680 (1:10000, LI-COR Biosciences, Cambridge, UK); goat anti-rat IR Dye 680 (1:10000, LI-COR); and donkey anti-rabbit IR Dye 800CW (1:10000, LI-COR). Membranes were washed again in PBS-T (four times for 5 minutes each time) and the fluorescent bands were detected using LI-COR’s Odyssey™ infrared imaging system.
Leukemia inhibitory factor ELISA
A total of 1 mL of supernatant was collected from each well of the six-well plates of primary mouse astrocyte cultures, and these samples were stored at −20°C. ELISA plates (96-well, Costar, Corning Life Sciences, Amsterdam, The Netherlands) were coated overnight at room temperature with 100 μl/well of primary antibody goat anti-LIF (AF449; 0.5 μg/mL, R&D Systems, Oxford, UK) diluted in 0.01 M PBS (pH 7.4). The following day, the plates were washed six times with wash buffer (0.25 M Tris–HCl pH 8, 0.15 M NaCl, 0.05% Tween-20) using an automated microplate washer and air dried (this step is repeated after each incubation step). Plates were subsequently incubated for 1 hour at room temperature with 200 μl/well of blocking buffer (0.01 M PBS, 2% BSA). After blocking, the plates were incubated with supernatants from astrocyte cultures (100 μl/well) for 2 hours at room temperature. Two dilutions (1:2 and 1:4) of each sample, diluted in incubation buffer (0.01 M PBS, 0.2% gelatin, 0.05% Tween-20), were made in triplicates. The plates were then incubated for 1 hour at room temperature with 100 μl/well of the detection antibody, biotinylated goat anti-LIF (BAF449; 0.05 μg/mL; R&D Systems, Oxford, UK) diluted in incubation buffer, followed by an incubation for 30 minutes at room temperature with 100 μl/well of Streptavidin-horseradish peroxidase (HRP) conjugate (1:8000, Sanquin Reagents, Amsterdam, The Netherlands). The plates were then incubated for 15 to 20 minutes at room temperature with 100 μl/well of TMB detection buffer (0.1 M acetate buffer, 0.1 M sodium-acetate, pH adjusted with 1 M citric acid (0.21 g/mL; dissolve 2 tablets of 3, 3′, 5, 5′-tetramethyl benzidinedihydrochloride in 11 mL of TMB buffer and add 2 μl of 30% H2O2)). Upon stable color formation the reactions were stopped by adding 100 μl/well of 1 M H2SO4. Absorbance of the samples was measured using VersaMax, a spectrophotometric ELISA plate reader, and SoftMax Pro software (Molecular Devices, CA, USA) at 450 nm, with a background correction at 575 nm. Recombinant mouse LIF (15 to 2000 pg/mL) was used to plot the standard curve.
Survival of cultured embryonic cortical neurons or cultured neonatal astrocytes against various experimental treatments was measured by the colorimetric MTT (3-(4,5-dimethylthiazol-2-yl-) 2,5-diphenyltetrazolium bromide) assay, as described previously . MTT solution (0.5 mg/mL final concentration) was added to cultured cells and incubated for 4 hours, after which, cells were lysed and MTT-formazan solubilized in dimethyl sulfoxide (DMSO) on an orbital shaker for 15 minutes. Optical density measure of each sample was determined using an automated ELISA reader - the Varioskan Flash spectral scanning multimode reader (Thermo Scientific, FL, USA) at 570 nm, with a background correction at 630 nm.
Immunocytochemistry and confocal microscopy
Astrocytes cultured on glass cover slips were fixed for 15 minutes in 4% paraformaldehyde. After several washes in PBS, the cells were blocked for 45 minutes with 5% normal goat serum (Vector Laboratories, Burlingame, CA, USA) in PBS containing 0.1% TritonX (Sigma, Zwijndrecht, The Netherlands). The cover slips were then incubated overnight at 4°C with rat anti-LIF primary antibody (5 μg/mL, R&D Systems, Oxford, UK) in combination with one of the following primary antibodies: rabbit anti-Rab11 (1:400, Zymed, San Francisco, CA, USA); rabbit anti-chromogranin A & B (1:100, Novus Biologicals, Cambridge, UK); mouse anti-clathrin (1:1000, Abcam, Cambridge, UK); rabbit anti-pHogrin C-terminal (1:100, kind gift of Professor J.C. Hutton (Denver, USA)) and rabbit anti-giantin (1:1000, Covance, Princeton, NJ, USA). The following day, cells were rinsed three times with PBS and incubated for 1 hour with the appropriate secondary antibodies: donkey anti-rat CY3 (1:500, Jackson ImmunoResearch Laboratories, Uden, The Netherlands); donkey anti-rabbit Alexa Fluor 488 (1:500, Molecular Probes, Leiden, The Netherlands)and donkey anti-mouse Alexa Fluor 488 (1:500, Molecular Probes). The cover slips were then rinsed with PBS and mounted on microscopic slide with Mowiol (Sigma, Zwijndrecht, The Netherlands) and analyzed with a Leica SP2 AOBS system (Leica Microsystems, Rijswijk, The Netherlands). Pictures were deconvoluted using the software Huygens Pro (SVI, Hilversum, The Netherlands). Primary antibody omission served as the control.
Statistical data analysis
The absolute data values were normalized to the control in order to allow multiple comparisons. Statistical analyses were performed by one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test, using the Statistical Package for the Social Sciences (SPSS, Chicago, IL, USA). In all cases, P values < 0.05 were considered statistically significant.
Glutamate-challenged cortical neurons induce LIF expression in cultured astrocytes through adenosine receptor activation
NECA-induced LIF expression and secretion levels in cultured mouse astrocytes is concentration- and time-dependent
NECA-induced LIF expression and secretion levels is dependent on adenosine A2B receptor activation
NECA-induced LIF expression and secretion levels in primary astrocytes are mediated through the Gq/11-PLC-PKC and MAPKs, but not through Gs-cAMP-PKA pathway.
Basal and NECA-induced LIF expression and secretion levels in primary astrocytes are dependent on ERK1/2- and p38- but not on JNK-MAPK activation
Basal and NECA-induced LIF expression and secretion levels in primary astrocytes are dependent on NF-κB activation
LIF secretion in primary astrocytes is constitutive and independent of NECA stimulation
LIF secretion in primary astrocytes is mediated through recycling endosomes
NECA-treated astrocytes induce LIF-mediated protection of cultured cortical neurons against excitotoxicity
We have previously shown that recombinant LIF protects neurons against glutamate-induced excitotoxicity . In this study, we investigated the mechanism by which astrocytes produce and release LIF. Here we show that glutamate-induced neuronal excitotoxicity leads to adenosine receptor-mediated increase in LIF mRNA expression in cultured cortical astrocytes. We demonstrate that the upregulation of LIF mRNA and protein is adenosine A2B receptor-dependent, and is mediated through Gq/11-PLC-PKC-MAPK-NF-κB signaling pathways. We furthermore show that LIF is transiting through the Golgi and is found in recycling endosomes rather than in LDCV. Finally, LIF produced by astrocytes can protect neurons against excitotoxicity.
It has been known for more than a decade that astrocytes are the major source for LIF in the CNS [18, 19, 59, 60]. However, the factors responsible for the regulation of LIF expression in these cells are still largely unknown. It is well known that stressed neurons release nucleotides such as ATP and adenosine [30, 61]. Recently, it was demonstrated that astrocytes increase LIF production and release in response to ATP receptor stimulation . In this study, the authors demonstrate that neurons during action potentials can secrete ATP, which triggers LIF production in astrocytes. This ATP-dependent upregulation of LIF by astrocytes is responsible for the promotion of oligodendrocyte-mediated myelination around neuronal axons. ATP is also known to be secreted by neurons during stressful conditions such as seizure, ischemia and hypoxia [26, 27]. However, when we blocked adenosine receptors with the non-selective antagonist caffeine, or with specific A2A/A2B receptor antagonists, the effect of glutamate-stressed neuronal supernatants on LIF expression in astrocytes was absent, suggesting that adenosine, but not ATP, is responsible for astrocytic LIF production during glutamate-induced neuronal stress. Thus, it might be hypothesized that depending on the CNS status, astrocytic LIF expression and secretion is differentially regulated; during normal neuronal activity and development ATP is involved whereas during neuronal insults, adenosine might enhance LIF secretion by astrocytes.
Several studies have demonstrated the involvement of adenosine A2B receptors in the regulation of IL-6 expression in various cell types in vitro[38, 47, 48, 62, 63] as well as in vivo, suggesting that A2B receptors might also be essential in the regulation of other IL-6-type cytokines. Our results show that adenosine-dependent LIF regulation is mediated through the A2B receptor, since no increase in LIF expression was found in cultured astrocytes from A2B receptor deficient mice. Instead NECA caused a down-regulation of LIF mRNA after 8 and 24 hours in these cells, indicating that knocking out A2B receptors may have unmasked an inhibitory effect on LIF mRNA expression of an unidentified adenosine receptor. Whether or not this might explain the very short-lived effect of NECA on LIF mRNA expression in wild-type astrocytes is at the moment unclear and a subject of future investigations. We furthermore demonstrated that A2B-mediated LIF expression is dependent on the PKC, but not the PKA pathway. These data are in line with the study of Aloisi and colleagues, which demonstrated that LIF modulation by pro-inflammatory cytokines in human astrocytes was mediated through PKC activation . Moreover, PKC has also been shown to be essential in IL-6 regulation [47, 48, 62, 65], revealing a prominent role for PKC in the signaling pathway controlling LIF gene expression.
MAPKs have been reported to be involved in adenosine A2B receptor-mediated regulation of IL-6 gene expression in astrocytoma cells . In our experiments, both basal as well as NECA-induced LIF gene expression and release in cultured astrocytes were inhibited by specific inhibitors of p38 and ERK1/2, but not JNK-MAPKs. In line with our findings, it has been shown that LIF expression in Schwann cells is mediated through PKC pathway-induced ERK1/2 activation . Furthermore, we show here that adenosine-dependent LIF expression in astrocytes is regulated through the NF-κB transcription factor. This observation is in line with several studies showing an NF-κB-dependent regulation of IL-6 gene by this transcription factor in several cell types [38, 50, 51, 65, 66]. It has been shown that NECA-induced NF-κB activation and the resultant IL-6 gene expression was abolished by inhibitors of MAPK pathways . In our study, preliminary observations indicate that NECA-induced activation of the NF-κB pathway is reduced by selective inhibitors of p38 and ERK1/2 pathways (data not shown), suggesting that these pathways might play as upstream mediators in NF-κB-dependent LIF expression in astrocytes.
Recent evidence indicates that, depending on the cell type, different secretory pathways are employed for cytokine release . For example, T cells use two different release mechanisms: IL-2 and IFN-γ are secreted at the immunological synapse whereas CCL3 and TNF-α are secreted multidirectionally, suggesting different secretory pathways . In neurons or neuron-like cells, secretory granules called LDCVs are the organelles used for the selective secretion of IL-6, TGF-β2 and CCL21 [53, 54, 69]. The same organelles are also used in immune cells such as mast cells and neutrophils . Here we show that LIF protein is transported through Golgi but its secretion by astrocytes is not mediated by secretory granules. Instead, LIF co-localizes with Rab11, a known marker of recycling endosomes [57, 58]. Moreover, we observed a partial co-localization of LIF with clathrin, which also associates with recycling endosomes where it is implicated in protein sorting . Recycling endosomes have now been shown to be responsible for cytokine secretion in several cell types. For example, IFN-γ and TNF-α secretion from natural killer cells require Rab11 . Recycling endosomes are also responsible for the constitutive secretion of IL-6 and TNF-α in macrophages . Further studies will be needed to better understand LIF sorting, trafficking and release by these vesicles.
Interestingly, our data indicate that LIF is constitutively released from astrocytes. Indeed constant levels of LIF were present in the supernatants of untreated astrocytes when measured by ELISA. Similar data were observed in human astrocyte cultures . Whether this observation is representative of the physiological behavior of astrocytes in vivo or is due to the culture conditions remains to be determined. We further show that by blocking the early secretory pathway with BFA, the LIF concentration in the culture supernatant was not increased upon NECA stimulation. The inhibitory effect of BFA indicates that LIF passes through the Golgi prior to its secretion, and thus does not follow non-conventional secretory pathways that by-pass the Golgi and is typically insensitive to BFA, which has recently been reported to be used by other cytokines . Importantly, the inhibitory effect of BFA suggests that NECA-stimulated release of LIF by astrocytes requires de novo LIF synthesis, and does not involve a ready-releasable post-Golgi pool of LIF.
It is now clear that one of the major roles of LIF is directed toward cell protection. Indeed, it has been shown that LIF is up regulated in astrocytes and neurons after cerebral ischemia  as well as in astrocytes after cortical brain injury , suggesting a role of LIF in neuronal repair or protection. In line with these data, treatment of rat with LIF prevented loss of motoneurons after peripheral nerve injury [73, 74] and protection of retinal ganglia cells was compromised in LIF knock-out mice after lens injury . Finally, LIF was shown to limit demyelination in an experimental autoimmune encephalomyelitis mouse model  and has become a prominent therapeutic candidate for multiple sclerosis . We have previously shown that LIF can protect cortical as well as hippocampal neurons against glutamate-induced excitotoxicity . Here we show that LIF coming from the supernatant of NECA-treated astrocytes has the same protective effect. Indeed, astrocytes produce several other cytokines and neurotrophic factors including IL-6, NGF, brain-derived neurotrophic factor, neurotrophin-3, S-100β protein and TGFβ , that might help neurons to cope with excitotoxic stress. Accordingly, conditioned media from astrocyte cultures protected cortical neurons against glutamate (data not shown). In order to confine the neuroprotective effect of astrocytic factors that are released in response to NECA treatment, we had to refresh astrocyte culture medium prior to NECA treatment and testing supernatant on glutamate-stressed neurons. This, together with LIF neutralization, indicates that LIF produced by astrocytes after adenosine receptor stimulation is necessary to witness neuronal protection. Our results provide further evidence for a role of adenosine in neuronal protection. Indeed, it has been shown that adenosine can protect neurons during hypoxia [76, 77], ischemia [78, 79] and excessive neuronal activity [80, 81]. This adenosine protection is often mediated through the A1 receptor subtype [82–84], but here we show that an indirect protection of adenosine through the stimulation of A2B receptor on astrocytes leading to LIF upregulation exists. This A2B receptor activation might be related to an anti-inflammatory process as observed previously by others [85–87].
We demonstrate a protective role of LIF against glutamate neurotoxicity and we provide clear evidence that adenosine is required for an increased production of LIF by astrocytes. These data further confirm a neuroprotective role of adenosine in the brain.
- A2B KO:
A2B receptor knock-out
central nervous system
ciliary neurotrophic factor
Dulbecco’s modified Eagle’s medium
Exchange Protein Activated by cAMP
extracellular signal-regulated kinase
fetal calf serum
Hank’s balanced salt solution
hydroxyethyl piperazineethanesulfonic acid
c-Jun N-terminal kinase
large dense-core vesicles
leukemia inhibitory factor
L-leucine methyl ester
Microtubule-associated protein 2
mitogen-activated protein kinase
nerve growth factor
nuclear transcription factor
Odyssey™ Blocking Buffer
phosphate-buffered saline plus 0.1 % Tween 20
protein kinase A
protein kinase C
recombinant mouse LIF
transforming growth factor β
This study was supported financially by the school of Behavioral and Cognitive Neurosciences (S. Moidunny) and by the Deutsche Forschungsgemeinschaft (K. Biber), grant number: FOR1336 and CA 115/5-4.
- Heinrich PC, Behrmann I, Haan S, Hermanns HM, Muller-Newen G, Schaper F: Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem J 2003, 374:1–20.View ArticlePubMedPubMed CentralGoogle Scholar
- Patterson PH: The emerging neuropoietic cytokine family: first CDF/LIF, CNTF and IL-6; next ONC, MGF, GCSF? Curr Opin Neurobiol 1992, 2:94–97.View ArticlePubMedGoogle Scholar
- Patterson PH: Leukemia inhibitory factor, a cytokine at the interface between neurobiology and immunology. Proc Natl Acad Sci USA 1994, 91:7833–7835.View ArticlePubMedPubMed CentralGoogle Scholar
- Kurek JB, Bower JJ, Romanella M, Koentgen F, Murphy M, Austin L: The role of leukemia inhibitory factor in skeletal muscle regeneration. Muscle Nerve 1997, 20:815–822.View ArticlePubMedGoogle Scholar
- Kwon YW, Abbondanzo SJ, Stewart CL, Gurney ME: Leukemia inhibitory factor influences the timing of programmed synapses withdrawal from neonatal muscles. J Neurobiol 1995, 28:35–50.View ArticlePubMedGoogle Scholar
- Akita S, Webster J, Ren SG, Takino H, Said J, Zand O, Melmed S: Human and murine pituitary expression of leukemia inhibitory factor. Novel intrapituitary regulation of adrenocorticotropin hormone synthesis and secretion. J Clin Invest 1995, 95:1288–1298.View ArticlePubMedPubMed CentralGoogle Scholar
- Chesnokova V, Auernhammer CJ, Melmed S: Murine leukemia inhibitory factor gene disruption attenuates the hypothalamo-pituitary-adrenal axis stress response. Endocrinology 1998, 139:2209–2216.PubMedGoogle Scholar
- Li M, Sendtner M, Smith A: Essential function of LIF receptor in motor neurons. Nature 1995, 378:724–727.View ArticlePubMedGoogle Scholar
- Murphy M, Reid K, Hilton DJ, Bartlett PF: Generation of sensory neurons is stimulated by leukemia inhibitory factor. Proc Natl Acad Sci USA 1991, 88:3498–3501.View ArticlePubMedPubMed CentralGoogle Scholar
- Mayer M, Bhakoo K, Noble M: Ciliary neurotrophic factor and leukemia inhibitory factor promote the generation, maturation and survival of oligodendrocytes in vitro. Development 1994, 120:143–153.PubMedGoogle Scholar
- Bugga L, Gadient RA, Kwan K, Stewart CL, Patterson PH: Analysis of neuronal and glial phenotypes in brains of mice deficient in leukemia inhibitory factor. J Neurobiol 1998, 36:509–524.View ArticlePubMedGoogle Scholar
- Leibinger M, Muller A, Andreadaki A, Hauk TG, Kirsch M, Fischer D: Neuroprotective and axon growth-promoting effects following inflammatory stimulation on mature retinal ganglion cells in mice depend on ciliary neurotrophic factor and leukemia inhibitory factor. J Neurosci 2009, 29:14334–14341.View ArticlePubMedGoogle Scholar
- Moidunny S, Dias RB, Wesseling E, Sekino Y, Boddeke HW, Sebastiao AM, Biber K: Interleukin-6-type cytokines in neuroprotection and neuromodulation: oncostatin M, but not leukemia inhibitory factor, requires neuronal adenosine A1 receptor function. J Neurochem 2010, 114:1667–1677.View ArticlePubMedGoogle Scholar
- Ueki Y, Wang J, Chollangi S, Ash JD: STAT3 activation in photoreceptors by leukemia inhibitory factor is associated with protection from light damage. J Neurochem 2008, 105:784–796.View ArticlePubMedGoogle Scholar
- Barres BA, Schmid R, Sendnter M, Raff MC: Multiple extracellular signals are required for long-term oligodendrocyte survival. Development 1993, 118:283–295.PubMedGoogle Scholar
- Butzkueven H, Zhang JG, Soilu-Hanninen M, Hochrein H, Chionh F, Shipham KA, Emery B, Turnley AM, Petratos S, Ernst M, Bartlett PF, Kilpatrick TJ: LIF receptor signaling limits immune-mediated demyelination by enhancing oligodendrocyte survival. Nat Med 2002, 8:613–619.View ArticlePubMedGoogle Scholar
- Slaets H, Hendriks JJ, Stinissen P, Kilpatrick TJ, Hellings N: Therapeutic potential of LIF in multiple sclerosis. Trends Mol Med 2010, 16:493–500.View ArticlePubMedGoogle Scholar
- Ishibashi T, Dakin KA, Stevens B, Lee PR, Kozlov SV, Stewart CL, Fields RD: Astrocytes promote myelination in response to electrical impulses. Neuron 2006, 49:823–832.View ArticlePubMedPubMed CentralGoogle Scholar
- Murphy GM, Song Y, Ong E, Lee YL, Schmidt KG, Bocchini V, Eng LF: Leukemia inhibitory factor mRNA is expressed in cortical astrocyte cultures but not in an immortalized microglial cell line. Neurosci Lett 1995, 184:48–51.View ArticlePubMedGoogle Scholar
- Slevin M, Krupinski J, Mitsios N, Perikleous C, Cuadrado E, Montaner J, Sanfeliu C, Luque A, Kumar S, Kumar P, Gaffney J: Leukaemia inhibitory factor is over-expressed by ischaemic brain tissue concomitant with reduced plasma expression following acute stroke. Eur J Neurol 2008, 15:29–37.PubMedGoogle Scholar
- Suzuki S, Tanaka K, Nogawa S, Ito D, Dembo T, Kosakai A, Fukuuchi Y: Immunohistochemical detection of leukemia inhibitory factor after focal cerebral ischemia in rats. J Cereb Blood Flow Metab 2000, 20:661–668.View ArticlePubMedGoogle Scholar
- Mashayekhi F, Salehi Z: Expression of leukemia inhibitory factor in the cerebrospinal fluid of patients with multiple sclerosis. J Clin Neurosci 2011, 18:951–954.View ArticlePubMedGoogle Scholar
- Soilu-Hanninen M, Broberg E, Roytta M, Mattila P, Rinne J, Hukkanen V: Expression of LIF and LIF receptor beta in Alzheimer’s and Parkinson’s diseases. Acta Neurol Scand 2010, 121:44–50.View ArticlePubMedGoogle Scholar
- Banner LR, Moayeri NN, Patterson PH: Leukemia inhibitory factor is expressed in astrocytes following cortical brain injury. Exp Neurol 1997, 147:1–9.View ArticlePubMedGoogle Scholar
- Yamakuni H, Kawaguchi N, Ohtani Y, Nakamura J, Katayama T, Nakagawa T, Minami M, Satoh M: ATP induces leukemia inhibitory factor mRNA in cultured rat astrocytes. J Neuroimmunol 2002, 129:43–50.View ArticlePubMedGoogle Scholar
- Cunha RA, Vizi ES, Ribeiro JA, Sebastiao AM: Preferential release of ATP and its extracellular catabolism as a source of adenosine upon high- but not low-frequency stimulation of rat hippocampal slices. J Neurochem 1996, 67:2180–2187.View ArticlePubMedGoogle Scholar
- Mitchell JB, Lupica CR, Dunwiddie TV: Activity-dependent release of endogenous adenosine modulates synaptic responses in the rat hippocampus. J Neurosci 1993, 13:3439–3447.PubMedGoogle Scholar
- Zimmermann H: Extracellular metabolism of ATP and other nucleotides. Naunyn Schmiedebergs Arch Pharmacol 2000, 362:299–309.View ArticlePubMedGoogle Scholar
- Berman RF, Fredholm BB, Aden U, O’Connor WT: Evidence for increased dorsal hippocampal adenosine release and metabolism during pharmacologically induced seizures in rats. Brain Res 2000, 872:44–53.View ArticlePubMedGoogle Scholar
- Lynch JJ, Alexander KM, Jarvis MF, Kowaluk EA: Inhibition of adenosine kinase during oxygen-glucose deprivation in rat cortical neuronal cultures. Neurosci Lett 1998, 252:207–210.View ArticlePubMedGoogle Scholar
- Parkinson FE, Xiong W: Stimulus- and cell-type-specific release of purines in cultured rat forebrain astrocytes and neurons. J Neurochem 2004, 88:1305–1312.View ArticlePubMedGoogle Scholar
- Parkinson FE, Xiong W, Zamzow CR: Astrocytes and neurons: different roles in regulating adenosine levels. Neurol Res 2005, 27:153–160.View ArticlePubMedGoogle Scholar
- von Lubitz DK: Adenosine and cerebral ischemia: therapeutic future or death of a brave concept? Eur J Pharmacol 1999, 371:85–102.View ArticlePubMedGoogle Scholar
- Hasko G, Pacher P, Vizi ES, Illes P: Adenosine receptor signaling in the brain immune system. Trends Pharmacol Sci 2005, 26:511–516.View ArticlePubMedPubMed CentralGoogle Scholar
- Ciccarelli R, Di Iorio P, Bruno V, Battaglia G, D’Alimonte I, D’Onofrio M, Nicoletti F, Caciagli F: Activation of A(1) adenosine or mGlu3 metabotropic glutamate receptors enhances the release of nerve growth factor and S-100beta protein from cultured astrocytes. Glia 1999, 27:275–281.View ArticlePubMedGoogle Scholar
- Fiebich BL, Biber K, Gyufko K, Berger M, Bauer J, van Calker D: Adenosine A2b receptors mediate an increase in interleukin (IL)-6 mRNA and IL-6 protein synthesis in human astroglioma cells. J Neurochem 1996, 66:1426–1431.View ArticlePubMedGoogle Scholar
- Heese K, Fiebich BL, Bauer J, Otten U: Nerve growth factor (NGF) expression in rat microglia is induced by adenosine A2a-receptors. Neurosci Lett 1997, 231:83–86.View ArticlePubMedGoogle Scholar
- Schwaninger M, Neher M, Viegas E, Schneider A, Spranger M: Stimulation of interleukin-6 secretion and gene transcription in primary astrocytes by adenosine. J Neurochem 1997, 69:1145–1150.View ArticlePubMedGoogle Scholar
- Wittendorp MC, Boddeke HW, Biber K: Adenosine A3 receptor-induced CCL2 synthesis in cultured mouse astrocytes. Glia 2004, 46:410–418.View ArticlePubMedGoogle Scholar
- Matos M, Augusto E, Oliveira CR, Agostinho P: Amyloid-beta peptide decreases glutamate uptake in cultured astrocytes: involvement of oxidative stress and mitogen-activated protein kinase cascades. Neuroscience 2008, 156:898–910.View ArticlePubMedGoogle Scholar
- Saura J: Microglial cells in astroglial cultures: a cautionary note. J Neuroinflammation 2007, 4:26.View ArticlePubMedPubMed CentralGoogle Scholar
- Hamby ME, Uliasz TF, Hewett SJ, Hewett JA: Characterization of an improved procedure for the removal of microglia from confluent monolayers of primary astrocytes. J Neurosci Methods 2006, 150:128–137.View ArticlePubMedGoogle Scholar
- Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods 2001, 25:402–408.View ArticlePubMedGoogle Scholar
- Mosmann T: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983, 65:55–63.View ArticlePubMedGoogle Scholar
- Feoktistov I, Biaggioni I: Adenosine A2B receptors. Pharmacol Rev 1997, 49:381–402.PubMedGoogle Scholar
- Fredholm BB, Irenius E, Kull B, Schulte G: Comparison of the potency of adenosine as an agonist at human adenosine receptors expressed in Chinese hamster ovary cells. Biochem Pharmacol 2001, 61:443–448.View ArticlePubMedGoogle Scholar
- Feng W, Song Y, Chen C, Lu ZZ, Zhang Y: Stimulation of adenosine A(2B) receptors induces interleukin-6 secretion in cardiac fibroblasts via the PKC-delta-P38 signalling pathway. Br J Pharmacol 2010, 159:1598–1607.View ArticlePubMedGoogle Scholar
- Fiebich BL, Akundi RS, Biber K, Hamke M, Schmidt C, Butcher RD, van Calker D, Willmroth F: IL-6 expression induced by adenosine A2b receptor stimulation in U373 MG cells depends on p38 mitogen activated kinase and protein kinase C. Neurochem Int 2005, 46:501–512.View ArticlePubMedGoogle Scholar
- Nagamoto-Combs K, Vaccariello SA, Zigmond RE: The levels of leukemia inhibitory factor mRNA in a Schwann cell line are regulated by multiple second messenger pathways. J Neurochem 1999, 72:1871–1881.View ArticlePubMedGoogle Scholar
- Libermann TA, Baltimore D: Activation of interleukin-6 gene expression through the NF-κB transcription factor. Mol Cell Biol 1990, 10:2327–2334.View ArticlePubMedPubMed CentralGoogle Scholar
- Spooren A, Kooijman R, Lintermans B, Van Craenenbroeck K, Vermeulen L, Haegeman G, Gerlo S: Cooperation of NFκB and CREB to induce synergistic IL-6 expression in astrocytes. Cell Signal 2010, 22:871–881.View ArticlePubMedGoogle Scholar
- Klausner RD, Donaldson JG, Lippincott-Schwartz J: Brefeldin A: insights into the control of membrane traffic and organelle structure. J Cell Biol 1992, 116:1071–1080.View ArticlePubMedGoogle Scholar
- Moller JC, Kruttgen A, Burmester R, Weis J, Oertel WH, Shooter EM: Release of interleukin-6 via the regulated secretory pathway in PC12 cells. Neurosci Lett 2006, 400:75–79.View ArticlePubMedGoogle Scholar
- Specht H, Peterziel H, Bajohrs M, Gerdes HH, Krieglstein K, Unsicker K: Transforming growth factor beta2 is released from PC12 cells via the regulated pathway of secretion. Mol Cell Neurosci 2003, 22:75–86.View ArticlePubMedGoogle Scholar
- McPherson PS: Proteomic analysis of clathrin-coated vesicles. Proteomics 2010, 10:4025–4039.View ArticlePubMedGoogle Scholar
- Deborde S, Perret E, Gravotta D, Deora A, Salvarezza S, Schreiner R, Rodriguez-Boulan E: Clathrin is a key regulator of basolateral polarity. Nature 2008, 452:719–723.View ArticlePubMedPubMed CentralGoogle Scholar
- van Ijzendoorn SC, Mostov KE, Hoekstra D: Role of rab proteins in epithelial membrane traffic. Int Rev Cytol 2003, 232:59–88.View ArticlePubMedGoogle Scholar
- van Ijzendoorn SC: Recycling endosomes. J Cell Sci 2006, 119:1679–1681.View ArticlePubMedGoogle Scholar
- Aloisi F, Rosa S, Testa U, Bonsi P, Russo G, Peschle C, Levi G: Regulation of leukemia inhibitory factor synthesis in cultured human astrocytes. J Immunol 1994, 152:5022–5031.PubMedGoogle Scholar
- Dallner C, Woods AG, Deller T, Kirsch M, Hofmann HD: CNTF and CNTF receptor alpha are constitutively expressed by astrocytes in the mouse brain. Glia 2002, 37:374–378.View ArticlePubMedGoogle Scholar
- Elliott MR, Chekeni FB, Trampont PC, Lazarowski ER, Kadl A, Walk SF, Park D, Woodson RI, Ostankovich M, Sharma P, Lysiak JJ, Harden TK, Leitinger N, Ravichandran KS: Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance. Nature 2009, 461:282–286.View ArticlePubMedPubMed CentralGoogle Scholar
- Rees DA, Lewis BM, Lewis MD, Francis K, Scanlon MF, Ham J: Adenosine-induced IL-6 expression in pituitary folliculostellate cells is mediated via A2b adenosine receptors coupled to PKC and p38 MAPK. Br J Pharmacol 2003, 140:764–772.View ArticlePubMedGoogle Scholar
- Ryzhov S, Zaynagetdinov R, Goldstein AE, Novitskiy SV, Blackburn MR, Biaggioni I, Feoktistov I: Effect of A2B adenosine receptor gene ablation on adenosine-dependent regulation of proinflammatory cytokines. J Pharmacol Exp Ther 2008, 324:694–700.View ArticlePubMedGoogle Scholar
- Vazquez JF, Clement HW, Sommer O, Schulz E, van Calker D: Local stimulation of the adenosine A2B receptors induces an increased release of IL-6 in mouse striatum: an in vivo microdialysis study. J Neurochem 2008, 105:904–909.View ArticlePubMedGoogle Scholar
- Kim MO, Kim MH, Lee SH, Suh HN, Lee YJ, Lee MY, Han HJ: 5'-N-ethylcarboxamide induces IL-6 expression via MAPKs and NF-κB activation through Akt, Ca(2+)/PKC, cAMP signaling pathways in mouse embryonic stem cells. J Cell Physiol 2009, 219:752–759.View ArticlePubMedGoogle Scholar
- Matsusaka T, Fujikawa K, Nishio Y, Mukaida N, Matsushima K, Kishimoto T, Akira S: Transcription factors NF-IL6 and NF-κB synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8. Proc Natl Acad Sci USA 1993, 90:10193–10197.View ArticlePubMedPubMed CentralGoogle Scholar
- Stow JL, Low PC, Offenhauser C, Sangermani D: Cytokine secretion in macrophages and other cells: pathways and mediators. Immunobiology 2009, 214:601–612.View ArticlePubMedGoogle Scholar
- Huse M, Lillemeier BF, Kuhns MS, Chen DS, Davis MM: T cells use two directionally distinct pathways for cytokine secretion. Nat Immunol 2006, 7:247–255.View ArticlePubMedGoogle Scholar
- de Jong EK, Vinet J, Stanulovic VS, Meijer M, Wesseling E, Sjollema K, Boddeke HW, Biber K: Expression, transport, and axonal sorting of neuronal CCL21 in large dense-core vesicles. FASEB J 2008, 22:4136–4145.View ArticlePubMedGoogle Scholar
- Reefman E, Kay JG, Wood SM, Offenhauser C, Brown DL, Roy S, Stanley AC, Low PC, Manderson AP, Stow JL: Cytokine secretion is distinct from secretion of cytotoxic granules in NK cells. J Immunol 2010, 184:4852–4862.View ArticlePubMedGoogle Scholar
- Manderson AP, Kay JG, Hammond LA, Brown DL, Stow JL: Subcompartments of the macrophage recycling endosome direct the differential secretion of IL-6 and TNFalpha. J Cell Biol 2007, 178:57–69.View ArticlePubMedPubMed CentralGoogle Scholar
- Nickel W, Rabouille C: Mechanisms of regulated unconventional protein secretion. Nat Rev Mol Cell Biol 2009, 10:148–155.View ArticlePubMedGoogle Scholar
- Cheema SS, Richards LJ, Murphy M, Bartlett PF: Leukaemia inhibitory factor rescues motoneurones from axotomy-induced cell death. Neuroreport 1994, 5:989–992.View ArticlePubMedGoogle Scholar
- Tham S, Dowsing B, Finkelstein D, Donato R, Cheema SS, Bartlett PF, Morrison WA: Leukemia inhibitory factor enhances the regeneration of transected rat sciatic nerve and the function of reinnervated muscle. J Neurosci Res 1997, 47:208–215.View ArticlePubMedGoogle Scholar
- Nakagawa T, Schwartz JP: Expression of neurotrophic factors and cytokines and their receptors on astrocytesin vivo.In Advances in Molecular and Cell Biology: Non-Neuronal Cells of the Nervous System: Function and Dysfunction Volume 31. Elsevier, Amsterdam; 2003:561–573.View ArticleGoogle Scholar
- Gribkoff VK, Bauman LA: Endogenous adenosine contributes to hypoxic synaptic depression in hippocampus from young and aged rats. J Neurophysiol 1992, 68:620–628.PubMedGoogle Scholar
- Fowler JC: Purine release and inhibition of synaptic transmission during hypoxia and hypoglycemia in rat hippocampal slices. Neurosci Lett 1993, 157:83–86.View ArticlePubMedGoogle Scholar
- Lloyd HG, Lindstrom K, Fredholm BB: Intracellular formation and release of adenosine from rat hippocampal slices evoked by electrical stimulation or energy depletion. Neurochem Int 1993, 23:173–185.View ArticlePubMedGoogle Scholar
- Latini S, Bordoni F, Corradetti R, Pepeu G, Pedata F: Effect of A2A adenosine receptor stimulation and antagonism on synaptic depression induced by in vitro ischaemia in rat hippocampal slices. Br J Pharmacol 1999, 128:1035–1044.View ArticlePubMedPubMed CentralGoogle Scholar
- Arvin B, Neville LF, Pan J, Roberts PJ: 2-chloroadenosine attenuates kainic acid-induced toxicity within the rat straitum: relationship to release of glutamate and Ca2+ influx. Br J Pharmacol 1989, 98:225–235.View ArticlePubMedPubMed CentralGoogle Scholar
- Lloyd HG, Perkins A, Spence I: Effect of magnesium on depression of the monosynaptic reflex induced by 2-chloroadenosine or hypoxia in the isolated spinal cord of neonatal rats. Neurosci Lett 1989, 101:175–181.View ArticlePubMedGoogle Scholar
- Pingle SC, Jajoo S, Mukherjea D, Sniderhan LF, Jhaveri KA, Marcuzzi A, Rybak LP, Maggirwar SB, Ramkumar V: Activation of the adenosine A1 receptor inhibits HIV-1 tat-induced apoptosis by reducing nuclear factor-kappaB activation and inducible nitric-oxide synthase. Mol Pharmacol 2007, 72:856–867.View ArticlePubMedGoogle Scholar
- Dunwiddie TV, Masino SA: The role and regulation of adenosine in the central nervous system. Annu Rev Neurosci 2001, 24:31–55.View ArticlePubMedGoogle Scholar
- Ramkumar V, Hallam DM, Nie Z: Adenosine, oxidative stress and cytoprotection. Jpn J Pharmacol 2001, 86:265–274.View ArticlePubMedGoogle Scholar
- Kuno A, Critz SD, Cui L, Solodushko V, Yang XM, Krahn T, Albrecht B, Philipp S, Cohen MV, Downey JM: Protein kinase C protects preconditioned rabbit hearts by increasing sensitivity of adenosine A2b-dependent signaling during early reperfusion. J Mol Cell Cardiol 2007, 43:262–271.View ArticlePubMedPubMed CentralGoogle Scholar
- Rosi S, McGann K, Hauss-Wegrzyniak B, Wenk GL: The influence of brain inflammation upon neuronal adenosine A2B receptors. J Neurochem 2003, 86:220–227.View ArticlePubMedGoogle Scholar
- Yang D, Zhang Y, Nguyen HG, Koupenova M, Chauhan AK, Makitalo M, Jones MR, St Hilaire C, Seldin DC, Toselli P, et al.: The A2B adenosine receptor protects against inflammation and excessive vascular adhesion. J Clin Invest 2006, 116:1913–1923.View ArticlePubMedPubMed CentralGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.