that adenosine A2b receptors participate in the regulation of the IL-6 gene in astrocytoma cells. Key Words: Inter- leukin-6âAstrocytesâAdenosineâAdenosine ...
Journal o~’Neuroche,notri Lippincott—Raven Puhhshers, Philadelphia © 1996 International Society for Neurochemistry
Adenosine A2b Receptors Mediate an Increase in Interleukin (IL)-6 mRNA and IL-6 Protein Synthesis in Human Astroglioma Cells *Bernd L. Fiebich, *~KnutBiber, *Kacin Gyufko, *Mathias Berger, *Joachim Bauer, and *Djetrjch van Calker *
Psychiatrische Klinik and
Biologi.s’ches Institut, Universitat Freiburg, Freiburg, Germany
Abstract: The cytokine interleukin (IL)-6 has recently been demonstrated to play a role in the pathology of Alzheimer’s disease (AD). The mechanisms leading to increased IL-6 levels in brains of AD patients are still unknown. Because in experimental animals ischemia increases both the level of cytokines and the extracellular concentrations of adenosine in the brain, we hypothesized that these two phenomena may be functionally connected and that adenosine might increase IL-6 gene expression in the brain. Here we show that the mixed A1 and A2 agonist 5’- (N-ethylcarboxamido) adenosine (NECA) induces an increase in IL-6 mRNA levels and protein synthesis in the human astrocytoma cell line U373 MG. The A1-specific agonists R-phenylisopropyladenosine and cyclopentyladenosine are much less potent, and the A2n-specific agonist CGS-21 680 shows only marginal effects. Increased levels of mRNA are already found within 30 mm after NECA treatment. The A2a-selective antagonists 8- (3-chlorostyryl ) caffeine and KF1 7837 [(E)-8-(3,4-dimethoxystyryl)-1 ,3-dipropyl-7-methylxanthine], which have also some antagonistic properties at A2b receptors, and the nonspecific adenosine antagonist 8-phenyltheophylline were equipotent at inhibiting the NECA-induced increase in lL-6 protein synthesis, whereas the specific A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine is much less potent. The results indicate that adenosine A2b receptors participate in the regulation of the IL-6 gene in astrocytoma cells. Key Words: Interleukin-6—Astrocytes—Adenosine—Adenosine A2b receptor—Alzheimer’s disease. J. Neurochem. 66, 1426—1431 (1996).
al., 1992). So far, little is known about the mechanisms that might induce IL-fl synthesis in AD brains, in rat brain, cytokine synthesis is increased by ischemia (for review, see Royston eta!., 1992), a condition that leads to a pronounced increase in content of extracellular adenosine (Rudolphi et al., 1992). In the brain, adenosine acts as a neuromodulator (see Williams, 1995). Adenosine receptors are expressed on both neurons and glial cells (Fredhoim and Altiok, 1994: Fredholm et a!., 1994) and were originally classified into the A1 and A2 subtypes by their differential effects on adenylyl cyclase (van Calker et al., 1979). Later on, several cyclic AMP (cAMP)independent adenosinergic effects were described (see Fredholm et a!., 1994). Four different adenosine receptor subtypes, all coupled to C proteins, have now been cloned (Fredholm et al., 1994). Little is known about the function of adenosine receptors on glial cells. Because glial cells are the main source of cytokines in the brain (Aloisi et al., 1992) and because levels of both cytokines and adenosine are increased following hypoxemia, we hypothesized that IL-6 gene expression in glial cells might be regulated by adenosine. Here we show that activation of adenosine A75 receptors induces the synthesis of lL-6 in the human astrocytoma cell line U373 MG. To our knowledge this is the first report that adenosine plays a regulatory role in the expression of a cytokine gene in the CNS.
Received September 8, 995; revised manuscript received November 28, 1995: accepted November 28. 1995. Address correspondence and reprint requests to Dr. B. L. Fiehich at Psychtalrtsche Kltntk der Universitht Freiburg, Neurochemischcs Labor. Hauptstrasse 5, D-79 104 Freiburg, Germany. Abbreviations used. AD. Alzheimer’s disease; cAMP, cyclic AMP; CPA, cyclopentyladenosine: CSC, 8-(3-chlorostyryl)caf-
The cytokine interleukin (IL)-6 regulates immune and inflammatory functions in the immune system and in the CNS (Frei and Fontana, 1989; Schöbitz et al., 1994). As we and others have found lL-6 in senile plaques in brains of Alzheimer’s disease (AD) patients, but not in brains of normal control persons (Woodetal., 1993; Huell etal., 1995), apathophysiological function of IL-6 in AD is postulated (Bauer et
fentc: DPCPX. 8-cyclopentyl- I .3-dipropyixanthine; IL, interleukin: KFI 7837, (E)-8-( 3.4-dimethoxystyryl )- I .3-dipropyl-7-methylxanthine; NECA, 5 ‘-(N-cthylcarhoxaniido)adenosine: R-PIA, R-phc’nylisopropyladenosine: 8-PT, 8-phenyltheophyl line.
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MATERIALS AND METHODS Materials Cyclopentyladenosine (CPA), CGS-2 1680, 8- ( 3-chlorostyryl ) caffeine (CSC), 5’-(N-ethylcarboxamido) adenosine (NECA), R-phenylisopropyladenosine (R-PIA), and 8cyclopentyl- I ,3-dipropylxanthine (DPCPX) were purchased from RBI (distributed by Biotrend, Köln, Germany), and 8-phenyitheophylline (8-PT) was obtained from Sigma (Deisenhofen, Germany). Stock solutions (10 mM) were prepared in dimethyl sulfoxide and stored at —20°C.Further dilutions were carried out in distilled water except that of CSC, which was prepared in dimethyl sulfoxide for all dilutions. KFI 7837 [(E) -8- (3,4-dimethoxystyryl) -1 ,3-dipropyl-7-methylxanthine] (Kyowa Hakko) was kindly provided by Boehringer Ingelheim KG (Germany) and dissolved in dimethyl sulfoxide for 10 mM stock solutions. Substances were diluted in culture medium as required before experiments. The IL-6 eDNA probe was kindly provided by Dr. T. Hirano (Institute for Molecular and Cellular Biology, Osaka University, Osaka, Japan). The eDNA coding for ~-actin was a generous gift from Dr. G. Finkenzeller (Institute for Tumor Biology, Freiburg, Germany).
Cell culture The human astrocytoma cell line U373 MG was obtained from the American Type Culture Collection (Rockville, MD, U.S.A.) and was grown in minimal essential medium-Earle’s medium (Seromed, Berlin, Germany) containing 10% fetal calf serum, L-glutamifle, antibiotics, vitamins, amino acids, and pyruvate. Cells were plated for RNA extraction in 10cm-diameter dishes (Falcon; l0~cells per dish in 10 ml of culture medium) and for protein analysis in six-well plates (Falcon; 2.5 X l0~cells per well in 2.5 ml of culture medium). Cultures were grown for 5—6 days at 37°C in 5% CO2. Medium was changed I day before treatment for RNA extraction and just before stimulation for IL-6 ELISAs, For incubation with the various compounds, the agents were added directly to the culture medium,
RNA extraction and northern analysis Total RNA was extracted using a RNA extraction kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocols. Total RNA (10 ~g per lane) was separated by agarose-formaldehyde gel electrophoresis, blotted onto positively charged nylon membranes (Pharmacia, Freiburg), and crosslinked by exposure to 120°Cfor 30 mm. The filters were prehybridized in 50% formamide, 0.25 M phosphate buffer (pH 7.2), 0,25 MNaCI, 10 mMEDTA, 200 p.g/ml of salmon sperm DNA, and 7% sodium dodecyl sulfate at 43°Cfor 2 Braunschweig, h.32P]dCTP The IL-6(Amersham, eDNA probe was labeledGermany) with 50 using pCi ofa [random priming kit from Boehringer (Mannheim, Germany). Unincorporated nucleotides were removed using a nucleotide removal kit from Qiagen. An overnight hybridization was performed at 43°Cusing the same buffer as for prehybridization. Membranes were washed in 2 >< saline— sodium citrate (SSC) containing 0.1% sodium dodecyl sulfate (3 >< 20 mm) at 60°Cand exposed to Kodak XAR film at —80°Cfor adequate intervals. For further hybridization, probes were removed by boiling the filter at 95°Cin distilled water.
IL-6 ELISA U373 MG cells were incubated for 24 h with adenosine agonists, whereas the antagonists were added 30 mm before
FIG. 1. IL-6 protein synthesis after treatment of U373 MG cells with CPA, R-PIA, CGS-21680, and NECA. U373 MG cells were incubated for 24 h with the agonists. Then the supernatants were harvested, and IL-6 protein synthesis was quantified using an IL-6 ELISA kit. Experiments with CGS 21680 were done separately. Data are mean ± SD (bars) values; SDs smaller than the symbol are not shown.
agonist treatment and also left for 24 h. Supernatants were then removed, centrifuged for 10 mm at 10,000 g, and stored at —80°C. For IL-6 ELISA (Immunotech, Hamburg, Germany), IL-6-containing supernatants were diluted in phosphate-buffered saline (1:50), and the lL-6 ELISA was carried out according to the manufacturer’s protocol. ELISA plates were shaken overnight at 4°C,washed five times with washing buffer, and then incubated for 30 mm with the substrate.
RESULTS Stimulation of U373 MG astrocytoma cells by the mixed adenosine A 1 and A2 agonist NECA led to a pronounced increase of IL-6 release into the culture medium. R-PIA, which is more selective for A1 receptors, was less potent, whereas the specific A1 agonist CPA had only a marginal effect. Stimulation with the specific A2~agonist CGS-21680 only led to a very low increase of IL-6 synthesis (Fig. I). Therefore, we focused our further studies on the effect of NECA. Northern analysis showed that IL-6 mRNA levels were dose-dependently increased by NECA. A plateau was reached at l0~ M NECA; higher concentrations (10 ~ M) did not further increase IL-6 transcript levels (Fig. 2A). A significant increase in IL-6 mRNA levels was already observed 30 mm after addition of NECA; J. NeuroeJie,n., Vol. 66, No. 4, 1996
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B. L. F1EB/~H ET AL.
FIG. 2. A: Northern blot analysis of U373 MG cells stimulated with different concentrations of NECA. U373 MG cells were stimulated with different concentrations of NECA for 4 h. Total RNA was then extracted, and 10 pg was used in northern blot analysis. The northern blot was hybridized with a cDNA probe coding for IL-6 (upper panel) or a cDNA coding for ~3-actinfor standardization (lower panel). B: Kinetics of the NECA-induced increase in IL-6 mRNA levels in U373 MG cells. 0373 MG cells were stimulated with 1 pM NECA for different intervals. Total RNA was then extracted, and 10 pg was used in northern blot analysis. After hybridization with a eDNA probe coding for IL-6 (upper panel), filters were hybridized with a /3-actin eDNA for standardization (lower panel).
a maximal induction was reached after 2 h, followed by a subsequent decrease (Fig. 2B). The selective A 2. antagonists CSC and KF17837 attenuated the NECA-induced increase of IL-6 protein synthesis, as did the nonspecific adenosine antagonist 8-PT. The adenosine A1 receptor-specific antagonist DPCPX was only effective at high concentrations (Fig. 3). Similarly, KF17837 but not DPCPX attenuated NECA-increased [L-6 mRNA levels (Fig. 4). A comparison of the NECA-increased EL-6 mRNA levels with other known stimuli of IL-6 mRNA expression is shown in Table I. The mRNA levels are increased l.2-ft)ld by cAMP (500 1iM), twofold by forskolin (10 pM), and fivefold by IL-l~compared with NECA (I pM). The IL-IN-induced mRNA expression is increased by NECA but not significantly more than in an additive manner (Table 1).
DISCUSSION In the present study we show that adenosine A25 receptors mediate an increase in the synthesis of lL-6
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in the astrocytoma cell line U373 MG, a well-established model system for studying IL-fl gene expression (Cadman et at., 1994; Fiebich et al., 1995; Lieb et al., 1996). This conclusion is based on the following findings: (a) The order of potency of adenosine agonists is NECA > R-PIA CPA, as would be expected for an A2 receptor-mediated effect (Fig. 1). (b) The specific A2~agonist CGS-21680 had almost no effect on IL-6 synthesis (Fig. I). (c) The potency of the selective A2~,antagonists CSC and KF17837 was similar to that of the nonspecific antagonist 8-PT, and the A1-selective antagonist DPCPX only marginally antagonized NECA responses (Fig. 3). The adenosine antagonist CSC has previously been shown to antagonize effects of the A25-specific agonist CGS-2 1680 (Jacobson et al., 1993) and to discriminate between A2~and A2h receptors (Daly and Jacobson, 1995), therefore suggesting that it is an A2-selective antagonist. Like CSC. KF17837 is described as a potent A2~antagonist (Nonaka et al., 1994a,h). The affinity of both compounds for the A21, receptors is much less pronounced. with IC25 values of 1.500 nM (Nonaka et al., 1994a,b) for KF17837 and 8,200 nM (Daly and Jacobson, 1995) for CSC. Our data (Fig. 3) show that both compounds inhibit the effect of NECA on 1L-6 protein synthesis with IC211 values of 1.000 nM, one order of magnitude greater
FIG. 3. IL-6 protein synthesis in U373 MG cells treated with NECA and different concentrations of the adenosine antagonists CSC, KF1 7837, DPCPX, and 8-PT. 0373 MG cells were incubated for 24 h with NECA; the antagonists were added 30 mm before NECA treatment. Supernatants were harvested, and IL6 protein synthesis was quantified using an lL-6 ELISA kit. Data are mean ± SD (bars) values given as percentages of the stimulation by NECA of IL-6 synthesis (defined as 100%, 24 ng/ml) in the absence of antagonists; SDs smaller than the symbol are not shown. CSC, KF17837, DPCPX, and 8-PT alone had no effect on IL-6 synthesis.
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ADENOSINE A,,, RECEPTOR-MEDIATED IL-6 GENE EXPRESSION
mRNA or to a so far unknown IL-fl splice product. Further investigations by western blot and PCR analysis should clarify this question. The rapid increase of IL-fl mRNA levels within 30 mm may be due to a
than the IC
511 expected for an A2, receptor but within the range expected for an A25 receptor-mediated response. Accordingly, the A2, receptor-specific agonist CGS2 1680 did not induce any IL-6 response in U373 MG cells. It is therefore concluded that the effect of adenosine agonists on IL-6 synthesis is mediated by A25 receptors. Indeed, there is evidence that astroglioma cells exclusively express adenosine A25 receptors (Fredholm and Altiok, 1994). The dose—response relationship of the NECA effect was identical in both an ELISA analysis of IL-fl protein synthesis and a northern blot analysis of IL-6 specific mRNA levels (Figs. I and 2). In the northern blot analysis a second band of lower molecular weight and unknown identity was observed only after NECA treatment (Fig. 2). This may represent nonspecific binding of the IL-6 cDNA probe to another NECA-induced
TABLE 1.
Comparison of the effects of NECA, dibutvrvl
cAMP, firskolin, and IL 1/3 on IL-6 mRNA levels in U373 MG cells Percentage NECA (1 pM)
100 12( 200 500 680
Dihutyryl cAMP (500 pM) Forskolin (10 pM) IL-l/3 (10 U/mI) IL-l/3 (It) U/nil) + NECA (I pM)
Data given are arbitrary units from northern blot analysis scanned with a Biometra system. The effect of NECA (I pM) was 989 ±22 arbitrary units (defined as 100%). Control levels of unstimulated cells were 110 ±It) arbitrary units. Blots were scanned four times, and the IL-b signals were standardiied with /3-aeOn.
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FIG. 4. Northern blot analysis of U373 MG cells stimulated with NECA and the adenosine antagonists DPCPX (A) and KF1 7837 (B). U373 MG cells were stimulated with 0.5 pM NECA. Antagonists were added 30 mm before incubation with NECA. Total RNA was then extracted, and 10 pg was subjected to northern blot analysis. The northern blot was hybridized with a eDNA probe coding for IL-6 (upper panel) or a eDNA coding for /3actin for standardization (lower panel).
regulation of IL-6 mRNA stability or to de novo mRNA synthesis. Further studies are needed to reveal if mRNA turnover or transcription is affected by NECA. It has been shown previously that varmous stimuli, including IL-I, substance P, vasoactive intestinal peptide, noradrenaline, and others, are capable of inducing IL-fl gene expression in primary astrocytes and U373 MG cells (Aloisi et al., 1992; Norris and Benveniste, 1993; Maimone et al., 1993). PKC and cAMP have been proposed as mediators in signal transduction leading to enhanced IL-fl gene expression in astrocytes (Grimaldi et al., 1994; Lieb et al., 1996). The only known signal transduction pathway activated by A2 receptors is the adenylyl cyclase system (Fredholm et a!., 1994). NECA stimulation of U373 MG cells induced an increased production of cAMP (data not shown). Therefore, cAMP is the most likely second messenger to mediate the induction of IL-6 synthesis by adenosine. Indeed, both IL-6 gene expression and protein synthesis can be increased by dibutyryl cAMP, forskolin, or /32-receptors, which are all known to activate the cAMP second messenger system (Maimone et al., 1993; Grimaldi et al., 1994). We have verified that dibutyryl cAMP and torskolin can also stimulate IL-6 protein synthesis in U373 MG cells (Table I). Both compounds increased IL-6 mRNA levels in a range comparable to those of NECA. IL-I/3, which is one of the strongest inducei’s of IL-fl mRNA expression in astrocytoma cells (Lieb et a!., 1996), increased IL6 mRNA levels five times as much as did NECA (Table I). In contrast to the situation in monocytes. where NECA enhances the IL-I-induced increase of IL-fl mRNA levels (Bouma et al., 1994), costimulation of U373 MG cells by NECA and IL-1/3 did not significantly enhance the increase of IL-fl mRNA levels. Only an approximately additive effect of both compounds
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B. L. FIEBICH ET AL.
was found (Table I). It has been reported that NECA, via A
2 receptors, inhibits the synthesis of tumor necrosis factor-a in monocytes and hepatocytes (Bouma et
al., 1994; Reinstein eta!., 1994). These results suggest that according to the cell type, the synthesis of cytokines could be modulated differentially by adenosine receptors. Most glial cell lines, as well as primary cultures of astroblasts from murine brain, express A25 receptors (Fredholm and Altiok, 1994; Fredholm et al., 1994). It is therefore assumed that the A25 receptors ubiquitously found in brain tissue are localized on astroglial cells. Little is known about the physiological function of these receptors. It has been reported that A2 receptors
play a role in astroglia proliferation (Rathbone et al., 1992; Hindley et a]., 1994). There is evidence suggesting that they are involved in the regulation of gly-
cogenolysis in astrocytes (Magistretti et a!., 1986). Another function of astroglial cells appears to be the regulation of brain-specific immunosurveiflance processes such as antigen presentation, expression of histocompatibility antigens, and synthesis of cytokines, including IL-fl and IL-! (Frei and Fontana. 1989). Our
results suggest that the regulation of these functions is in fact one important role of adenosine receptors on astroglial cells. The finding that signaling via astrocyte A25 receptors is modulated by IL-I (Fredhoim and
Altiok, 1994) is in line with this proposal. However, such a role for astroglial cells has been largely deduced from data obtained from cell lines or primary cultured glioblasts. The validity of these in vitro findings for the function of glial cells in vivo remains to be established. Induction of IL-fl synthesis by adenosine, if it also
occurs in mature astrocytes in vivo, would provide a unifying explanation for several hitherto disparate findings in AD. Neuronal damage induced by head injury is a well-known risk factor for AD (Mortimer, 1991). Conditions of ischemia and hypoxia (which are often superimposed on other pathological features of traumatic brain injury) cause a large increase in the concentration of extracellular adenosine, which can
protect neurons from excessive overstimulation and subsequent death (Rudolphi et al., 1992). Our results suggest that adenosine released under these conditions
could also activate, via A25 receptors, adjacent astrocytes to produce 1L-6 and perhaps also other factors that can induce increased survival and/or regeneration of neurons (Hama et al., 1989). The recent finding that hypoxia indeed induces IL-6 synthesis in rat astrocytes (Maeda et a!., 1994) corroborates this assumption. However, although advantageous under certain circumstances, this mechanism might also trigger an exaggerated inflammatory response that would result in detrimental effects for the brain (Bauer et al., 1991. 1992). In addition to astrocytes, microglia activation appears to participate also in the pathological processes leading to AD (for review, see McGeer et al., 1993). In line with this assumption, we have recently shown
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that adenosine agonists also stimulate microglia proliferation (Gebicke-Härter et al., 1996). In conclusion, our results suggest that an increase in the concentration of extracellular adenosine might induce increased IL-6 mRNA levels in astrocytes. This
may provide a causal link among pathological conditions like brain trauma (a risk factor of AD), hypoxia as a consequence of amyloid angiopathy, and enhanced glial expression of IL-6. a cytokine possibly involved in the pathophysiology of AD.
Acknowledgment: We would like to thank Dr. A. PopaWagner for discussing the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 364/
A5, SFB 505/BI, and grants Ba 10511640 and Ca 115/51) and the Fritz-Thyssen-Stiftung (grant 1993/1-69),
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