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Keywords:

  • ATP;
  • IL-6;
  • microglia;
  • mitogen-activated protein kinase

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Microglia play various important roles in the CNS via the synthesis of cytokines. The ATP-evoked production of interleukin-6 (IL-6) and its intracellular signals were examined using a mouse microglial cell line, MG-5. ATP, but not its metabolites, produced IL-6 in a concentration-dependent manner. Although ATP activated two mitogen-activated protein kinases, i.e. p38 and extracellular signal-regulated protein kinase, only p38 was involved in the IL-6 induction. However, the activation of p38 was not sufficient for the IL-6 induction because 2′- and 3′-O-(4-benzoylbenzoyl) ATP, an agonist to P2X7 receptors, failed to produce IL-6 despite the fact that it activated p38. Unlike in other cytokines in microglial cells, P2Y rather than P2X7 receptors seem to have a major role in the IL-6 production by the cells. The ATP-evoked IL-6 production was attenuated by Gö6976, an inhibitor of Ca2+-dependent protein kinase C (PKC). The P2Y receptor responsible for these responses was insensitive to pertussis toxin (PTX) and was linked to phospholipase C. Taken together, ATP acting on PTX-insensitive P2Y receptors activates p38 and Ca2+-dependent PKC, thereby resulting in the mRNA expression and release of IL-6 in MG-5. This is a novel pathway for the induction of cytokines in microglia.

Abbreviations used
ADP

adenosine 5′-diphosphate

ATPγS

adenosine 5′-O-(3-thiotriphosphate)

BzATP

2′- and 3′-O-(4-benzoylbenzoyl) adenosine 5′-triphosphate

[Ca2+]i

intracellular Ca2+ concentration

DMEM

Dulbecco's modified Eagle's medium

ERK

extracellular signal-regulated protein kinase

FDA

fluorescencin diacetate

IL-1β

interleukine-1β

IL-6

interleukine-6

InsP3

inositol 1, 4, 5-trisphosphate

LPS

lipopollysaccharide

MAP kinase

mitogen activated protein kinase

NEM

N-ethylmaleimide

oATP

oxdized ATP

OD

optical density

PI

propidium iodide

PLC

phospholipase C

PKC

protein kinase C

PTX

pertussis toxin

TNF-α

tumor necrosis factor-α

UTP

uridine 5′-triphosphate.

There is increasing evidence that microglia have crucial roles in the maintenance of neuronal homeostasis in the CNS (Nakajima and Kohsaka 1993; Verkhratsky and Kettenmann 1996). The most characteristic feature of microglia is their rapid activation in response to pathological events including apoptosis, neurodegeneration and inflammation in the CNS. Injured cells can release or leak large amounts of ATP into the extracellular environment. Such extracellular ATP seems to be a key molecule for triggering microglial responses to pathological events, initiating and maintaining reactive microglia. In fact, ATP can exert mitogenic and morphogenic effects on glial cells (Neary et al. 1996a; Abbracchio et al. 1999; Brambilla et al. 1999). ATP can even cause microglial chemotaxis (Honda et al. 2001), which may underlie the microglial accumulation in the damaged brain region.

Microglia possess several functional receptors for ATP, i.e. metabotropic P2Y receptors and ionotropic P2X receptors such as P2X7 receptors (Ferrari et al. 1996; Neary et al. 1996a; Verkhratsky and Kettenmann 1996). ATP acting on these P2 receptors in microglia evokes many kinds of cellular responses such as activation of inward currents (Nörenberg et al. 1994), an increase in the intracellular Ca2+ concentration ([Ca2+]i) (Walz et al. 1993; Toescuet et al. 1998; McLarnon et al. 1999Möller et al. 2000), and phosphorylation of mitogen-activated protein (MAP) kinases (Gao et al. 1999; Neary and Zhu 1994; Soltoff et al. 1998; Neary et al. 1999). Some of these responses may be closely related to microglial activation.

Activated microglia have a dual-regulatory function to maintain or facilitate tissue homeostasis in the CNS. They remove dead cells or dangerous debris by releasing toxic factors and by phagocytosis, whereas they also repair injured cells by releasing neurotrophic factors (Nakajima and Kohsaka 1993; Inoue et al. 1998a). With regard to such microglial secretory responses, recent interest has focused on the P2X7 receptor because its activation can regulate the release of several important molecules such as cytokines and plasminogen in microglia. ATP evokes the release of interleukine-1β (IL-1β) (Ferrari et al. 1997a), tumor necrosis factor-α (TNF-α) (Hide et al. 2000; Morigiwa et al. 2000), and plasminogen (Inoue et al. 1998a,b) from microglia via P2X7 receptors. The stimulation of P2X7 receptors causes an elevation in [Ca2+]i or activation of MAP kinases, resulting in the induction of these molecules (Inoue et al. 1998b; Hide et al. 2000). Microglia possess other receptors for ATP such as P2Y receptors which can be activated simultaneously by released ATP. However, there have been few reports about P2Y receptor-mediated signals, much less about the induction of cytokines in relation to these P2Y receptors in microglia.

In the present study, we focused on IL-6, and investigated the ATP-evoked production of IL-6 and the mechanisms underlying its induction in a microglial cell line, MG-5. Several actions of IL-6 have been described in the brain or in neuron and glia in vitro, including some contradictory or even opposing effects (Gadient and Otten 1997). While IL-6 regulates neuronal survival and differentiation (Umegaki et al. 1996; März et al. 1997, 1998; Hirota et al. 1996) and has anti-inflammatory functions (Oh et al. 1998), other reports suggest that IL-6 is detrimental and contributes to the pathophysiology associated with CNS disorders (Campbell et al. 1993). Although the exact role of IL-6 in the CNS has not been clarified, it is known that this cytokine is up-regulated in many CNS disorders (Gruol and Nelson 1997; Hays 1998; Zhao and Schwartz 1998; Van Wagoner and Benveniste 1999). We demonstrate here that ATP stimulates the de novo synthesis of IL-6 through pathways mediated by both p38 and Ca2+-dependent protein kinase C (PKC) in the cells. The P2 receptors responsible for these responses are phospholipase C (PLC)-linked P2Y receptors which are insensitive to pertussis toxin (PTX). This is a novel pathway for the induction of cytokines in microglial cells.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Cell culture

The mouse microglial cell line, MG-5 were isolated from the brain of newborn p53 deficient mice as described previously by Ohsawa et al. (1997). MG-5 were grown in astrocyte-conditioned medium that is obtained from the supernatant of astrocytes cultured overnight in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum, 10 U/mL penicillin and 10 mg/mL streptomycin.

Measurement of IL-6 release

MG-5 were cultured in 24 well plates (8 × 104 cells/0.4 mL/well) and stimulated with ATP, and then the supernatant was collected for the assay of IL-6. IL-6 in the supernatant was measured by Cytoscreen mouse IL-6 immunoassay kit (Biosource International, Camarillo, CA, USA). The assay was performed according to the manufacturer's protocol. All reagents used were checked for endotoxin contamination. Moreover, the ATP-evoked IL-6 release was observed even in the presence of polymixin B (109.6% of ATP alone), suggesting that the effect of contaminating endotoxin on the IL-6 secretion would be negligible.

Quantitative RT-PCR of IL-6 mRNA

MG-5 were prepared in 35 mm dishes (4 × 105 cells/2 mL/dish) and stimulated with ATP for 6 h. At the end of stimulation, the cells were directly lysed with 0.5 mL of RNA STAT-60 (Tel-Test ‘B’ Inc., Friendswood, TX, USA) and total RNA was isolated and purified according to the manufacturer's instructions. Reverse transcription (RT) was performed with 3 µg of total RNA using M-MLV reverse transcriptase (Life Technologies, Grand Island, NY, USA). The number of IL-6 gene copies was measured by a Cytoxpress Mouse IL-6 Quantitative PCR Detection Kit (Biosource International, Camarillo, CA, USA). The RT product (5 μL) was added to the reaction mixture containing 1 × PCR buffer (10 mm Tris-HCl, pH 8.3, 50 mm KCl), 1.5 mm MgCl2, 0.2 mm dNTPs, 2.5 units of Taq polymerase, 50 pmol of the IL-6 primer pair and 2000 copies of the Internal Calibration Standard (ICS). After PCR amplification, the products were detected by agarose gel electrophoresis and quantified in 96 well microplates with the kit. Each PCR product (10 µL) was subjected to electrophoresis on 2% agarose gel, which was then stained with ethidium bromide and photographed. The number of copies of IL-6 in each PCR reaction was calculated from the ratio of the total optical density (OD) for the IL-6 specific well to the total OD for the ICS well and the input copy number of the ICS.

MAP kinase phosphorylation assay

MG-5 were prepared in 35 mm dishes (3 × 105 cells/1.5 mL/dish). After ATP-stimulation, the cells were lysed, and the lysates were resolved with 12.5% SDS–PAGE gels and transferred to nitrocellulose membranes. The membranes were blocked for 1 h in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) and 5% non-fat dry milk at 25°C. Then the membranes were incubated with primary antibody dilution buffer (1 : 1000 dilution into TBS-T containing 5% BSA) overnight at 4°C. After three washes, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit antibody (1: 2000 dilution into TBS-T containing 5% non-fat dry milk) for 1 h at 25°C. The membranes were washed three times, and the MAP kinases were visualized by chemiluminescence. The anti-phospho-MAP kinase antibody specifically detected only the activated, phosphorylated form of the MAP kinases.

Measurement of intracellular Ca2+ concentration ([Ca2+]i) in single cells

MG-5 were cultured in poly-l-lysine and collagen-coated glass coverslip at a density of 2 × 105 cells/mL. The changes in [Ca2+]i in single cells were measured by the fura-2 method as described previously (Koizumi et al. 1994) with minor modifications. The cells were washed with DMEM and incubated with 3 µm fura-2 acetoxymethylester (fura-2 AM; Dojindo, Kumamoto, Japan) for 45 min in DMEM. Fura-2-loaded cells were placed on a fluorescence-image microscope and perfused with medium at room temperature (22–25°C). Drugs were dissolved in DMEM and applied by superfusion. The fura-2 fluorescence was measured with excitation at 340 nm and 360 nm, and at an emission wavelength of 510 ± 20 nm.

Detection of mRNAs for P2 receptors

The MG-5 were directly lysed with 0.5 mL of RNA STAT-60 (Tel-Test ‘B’ Inc.) and total RNA was isolated. Reverse transcription was performed with 1 µg of total RNA using M-MLV reverse transcriptase. The RT product (1 µL) was added to the reaction mixture containing 1 × PCR buffer (10 mm Tris-HCl, pH 8.3, 50 mm KCl), 1.5 mm MgCl2, 0.2 mm dNTPs, 2.5 units of Taq polymerase, and P2Y1, P2Y2, P2Y4, P2Y6 and P2X7 receptors specific primers according to the nucleotide sequence as follows: P2Y1, 5′-CTGATCTTGGGCTGTTATGG-3′ forward, and 5′-GCT GTTGAGACTTGCTAGAC-3′ reverse; P2Y2, 5′-GGTTTATTAC TACGCCCAGG-3′ forward, and 5′-AAGGAGTAATAGAGGG TGCG-3′ reverse; P2Y4, 5′-CCTCGTCTACTACTATGCTGCC-3′ forward, and 5′-CACCATGACTGCCGAACTGAAG-3′ reverse; P2Y6, 5′-GTGGTATGTGGAGTCGTTTG-3′ forward, and 5′-CT GTAGGAGATCGTGTGGTT-3′ reverse; P2X7, 5′-TAGTACA CGGCATCTTCGAC-3′ forward, and 5′-CTGAACTGCCACCT CTGTAA-3′ reverse. After PCR amplification, the products were analyzed by electrophoresis on agarose gel with ethidium bromide.

Reagents

Reagents were obtained from the following sources: MAP kinases inhibitors PD 98059, SB 203580 and Gö6976 were from Calbiochem (La Jolla, CA, USA); ATP, adenosine 5′-O-(3-thiotriphosphate) (ATPγS), adenosine, adenosine 5′-diphosphate (ADP), uridine 5′-triphosphate (UTP), 2′- and 3′-O-(4-benzoylbenzoyl) adenosine 5′-triphosphate (BzATP) and bovine serum albumin (BSA), N-ethylmaleimide (NEM), aminophylline, PTX were from Sigma Chemical Co. (St Louis, MO, USA); Suramin was from Wako Pure Chemical Industries, Ltd. (Osaka, Japan); 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethylester (BAPTA-AM) was from Molecular Probes (Eugene, OR, USA); AmpliTaq DNA polymerase set for PCR was purchased from Perkin Elmer (Branchburg, NJ, USA); Phosphoplus p38 MAP kinase (Thr180/Tyr182) antibody, Phosphoplus P44/42 MAP kinase (Thr202/Tyr204) antibody and Phosphoplus SAPK/JNK MAP kinase (Thr183/Tyr185) antibody were from New England Bio Laboratories (Beverly, MA). The AR-C67085 was a gift from AstraZeneca (Loughborough, Leicestershire, UK). All materials for cell culture were from Life Technologies (Grand Island, NY, USA). Where possible, analytical quality reagents were used.

Data analysis and statistics

All results are expressed as the mean ± SEM. A statistical analysis was performed using Student's t-test, and differences were considered to be significant at p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Extracellular ATP triggers gene expression and release of IL-6 in MG-5

The effects of ATP and its analogues on the mRNA expression and release of IL-6 were investigated in MG-5. Figure 1(a) shows the time-course of the release of IL-6 evoked by ATP, ATPγS, ADP, adenosine, and UTP. The release of IL-6 showed a very slow time-course and the release of IL-6 was detected after incubation with ATP (1000 µm) for more than 6 h (Fig. 1a). This raises the possibility that metabolites of ATP such as ADP or adenosine might be involved in the release. However, neither ADP nor adenosine at 1000 µm stimulated the release of IL-6 (Fig. 1a). In addition, ATPγS, a non-hydrolysable analogue, induced a significant release of IL-6. Thus, ATP, but not its metabolites, appeared to be responsible for stimulating the release of IL-6. At 24 h the ATP produced the release of IL-6 in a concentration-dependent manner over a concentration range of 10–1000 µm(Fig. 1b). Higher ATP concentrations (3 and 5 mm) did not produce further IL-6 induction (at 3 mm, 34.1% of 1 mm ATP; at 5 mm, 0% of 1 mm ATP). UTP did not evoke IL-6 release (Fig. 1a).

image

Figure 1. Production of IL-6 by ATP in MG-5. (a) the time-course of the release of IL-6 in the cells. ATP, ●; ATPγS, ○; ADP, ▴; adenosine, ▵; UTP, ×. The cells were treated with 1 mm agonists for the times indicated. After stimulation of the cells for an appropriate period, the supernatant in each well was collected, and the released IL-6 was measured by ELISA as described in Materials and methods. Data show mean ± SEM of three independent experiments. (b) ATP stimulated the release of IL-6 in a concentration-dependent manner in MG-5. Twenty-four hours after treatment of the cells with various concentrations of ATP, the supernatant was collected for the IL-6 assay. Data show mean ± SEM of four independent experiments. (c) Induction of IL-6 mRNA evoked by ATP in MG-5. Cells were incubated with and without ATP for 6 h, and the IL-6 gene number was calculated as described in Materials and methods. Data were normalized by the control. Values show mean ± SEM of 14 independent experiments. Inset shows an image of the agarose gel electrophoresis. No significant difference could be observed in the expression of GAPDH mRNA among the experiments. The bands at 243 base pairs indicate IL-6 amplicons. Asterisks show significant difference from the response of basal (***p < 0.001). (d) Effect of suramin (100, 1000 µm, + Sur) and aminophylline (100 µm, + Aminop) on the ATP-evoked IL-6 mRNA expression. The cells were treated with each antagonist for 10 min, and then stimulated with 1 mm ATP for 6 h. After treatment of the cells with ATP in the presence or absence of the antagonists for 6 h, IL-6 mRNA expression was measured by quantitative RT-PCR as described in Materials and methods. Data were normalized by the control. Values show mean ± SEM of four independent experiments. Asterisk shows significant difference from the response evoked by ATP alone (**p < 0.01, ***p < 0.001).

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Next, we measured the expression of IL-6 mRNA in MG-5 by a competitive RT-PCR. The maximal IL-6 mRNA expression was detected at 6 h after the initial stimulation with 1000 µm ATP. As shown in Fig. 1(c), ATP at 6 h induced an approximately 8-fold increase in the IL-6 mRNA, which was significantly inhibited by suramin in a concentration-dependent manner (100 µm, 44.6 ± 6.1% of ATP alone (p < 0.01); 1000 µm, 5.6 ± 6.9% of ATP alone (p < 0.001) (Fig. 1d). Aminophylline (100 µm), a non-selective antagonist to adenosine receptors, did not affect the ATP-evoked IL-6 gene expression (Fig. 1d, +Aminop), which was well in accord with the result that adenosine did not produce IL-6 release. These results indicated that ATP stimulates the de novo synthesis of IL-6 via a P2 receptor–mediated pathway, leading to the subsequent release of IL-6.

After 24 h-incubation with ATP, the cell viability was determined by simultaneous staining with fluorescencin diacetate (FDA) and propidium iodide (PI) (Jones and Senft 1985). Viable cells hydrolyse FDA by intracellular esterases and become bright green, while non-viable cells are labelled by PI and become red. Almost all of the control cells and the ATP-treated cells showed bright green fluorescence with no significant difference in the staining between them, suggesting that the cells incubated with ATP for 24 h were alive (data not shown).

Extracellular ATP triggers p38 and extracellular signal-regulated protein kinase (ERK) 1/2 phosphorylation in MG-5

To elucidate the ATP-evoked intracellular signaling cascade, we focused on the activation of three types of MAP kinases, ERK1/2, p38, and jun N-terminal kinase (JNK), upon stimulation with ATP as these MAP kinases have been reported to be important for IL-6 gene expression in several cells (Berghe et al. 1998; Tuyt et al. 1999). p38 was rapidly phosphorylated by ATP (1000 µm) in a 1-min incubation period, and the phosphorylation remained at nearly the maximal level until 10 min after the stimulation. The phosphorylation was observed even at 60 min after the initial stimulation (Fig. 2a). Although ERK1/2 was also phosphorylated by ATP, the time-course was slower than that of p38 and its phosphorylation was detected 30 min after the ATP-stimulation (Fig. 2a). ATPγS phosphorylated p38 and ERK1/2 in a manner similar to that of ATP (data not shown). The ATP activated both p38 and ERK1/2 in a concentration-dependent manner (Fig. 2b). JNK was not activated by ATP (data not shown).

image

Figure 2. Phosphorylation of p38 and ERK1/2 MAP kinases in MG-5. (a) ATP activated p38 and ERK1/2 MAP kinases in the cells. The cells were treated with 1 mm ATP for the times indicated (0–120 min), lysed and then the lysates containing equivalent amounts of protein were subjected to SDS–PAGE. Immunoblots were probed with antibodies that recognize only the phosphorylated form of p38 (a, upper panel), ERK1/2 (a, third panel). For comparison, the total amounts of each MAPK are shown in the bottom of each panel. Upon stimulation with ATP, the phosphorylation of p38 and ERK1/2 peaked at 1 and 30 min, respectively. (b) ATP activated p38 and ERK1/2 MAPK in a concentration dependent manner in MG-5. The assay of phosphorylation was performed at 1 min for p38 and at 30 min for ERK1/2 after treatment with various concentrations of ATP. (c) Depicts the time-courses of the phosphorylation of p38 and ERK1/2 MAPK evoked by ADP (1 mm). These are results from a typical experiment. At least three such independent experiments were performed and similar results were obtained. Asterisks indicate phosphorylated form.

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ADP (1000 µm) also activated these MAP kinases (Fig. 2c), though it did not stimulate the release of IL-6 (Fig. 1a). Especially, the phosphorylation of ERK1/2 by ADP was remarkable, i.e. the phosphorylation was much faster and stronger than that by ATP. The ADP-evoked phosphorylation of ERK1/2 peaked at 1 min, whereas the ATP-evoked one could be observed at 30 min after stimulation. ADP also triggered the activation of p38, but its phosphorylation by ADP was transient and much less potent than that by ATP. Adenosine activated neither p38 nor ERK1/2 (data not shown). Neither ADP nor adenosine activated JNK (data not shown). No significant difference was observed in the total amounts of MAP kinases among the experiments.

Involvement of p38 activation in the ATP-evoked IL-6 production in MG-5

To determine whether the MAP kinase cascade is involved in the ATP-evoked IL-6 release, we investigated the effects of SB203580 and PD98059, inhibitors of p38 and ERK1/2, respectively, on the ATP-evoked IL-6 production in MG-5. As shown in Fig. 3(b), the ATP-evoked IL-6 release was inhibited by SB203580 (25 µm; +SB, p < 0.01) but not by PD98059 (25 µm; + PD). SB203580 also suppressed the ATP-evoked IL-6 mRNA induction (p < 0.05) but PD98059 did not (Fig. 3c). Both PD98059 and SB203580 had no effect on the basal release of IL-6 (data not shown). The specificity of these MAP kinase inhibitors was confirmed by the results that SB203580 selectively inhibited the ATP-evoked phosphorylation of p38 without affecting the activation of ERK1/2, and PD98059 inhibited only the phosphorylation of ERK1/2 (Fig. 3a). Thus, it is suggested that activation of p38 is essential for the ATP-evoked IL-6 production.

image

Figure 3. Effects of MAP kinase inhibitors on the ATP-evoked release of IL-6 and expression of IL-6 gene in MG-5 cells. (a) the ATP-evoked p38 and ERK1/2 MAP kinase activities were selectively inhibited by SB203580 (SB) and PD98059 (PD), respectively. The cells were treated with 25 µm PD98059 or SB203580 for 30 min, and then stimulated with 1 mm ATP for 1min (p38) and 30 min (ERK1/2) with and without MAP kinase inhibitors. These are results from a typical experiment. At least three such independent experiments were performed and similar results were obtained. SB203580 but not PD98059 inhibited the ATP-evoked IL-6 release (b) and IL-6 mRNA gene expression (c) in the cells. (b) After treatment of the cells with ATP in the presence or absence of MAP kinase inhibitors for 24 h, the supernatant in each well was collected, and released IL-6 was measured by immunoassay as described in Materials and methods. Data were normalized by the responses evoked by ATP alone. Values show mean ± SEM of four independent experiments. (c) After treatment of the cells with ATP in the presence or absence of MAP kinase inhibitors for 6 h, IL-6 mRNA expression was measured by quantitative RT-PCR as described in Materials and methods. Data were normalized by the control. Values show mean ± SEM of three–four independent experiments. The inset shows an image of the agarose gel electrophoresis. No significant difference could be observed in the expression of GAPDH mRNA among the experiments. The bands at 243 base pairs indicate IL-6 amplicons. Statistical significance is shown as *p < 0.05 and **p < 0.01 compared with control (ATP alone).

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The phoshorylation by BzATP of p38 failed to produce IL-6 in MG-5

The pathway(s) mediated by p38 was necessary for the ATP-evoked IL-6 production in MG-5 cells. We next examined the ATP-evoked changes in the [Ca2+]i and Ca2+ dependency of the ATP-evoked p38 activation. When extracellular Ca2+ was removed, the p38 phosphorylation was dramatically inhibited (Fig. 4a2). BAPTA-AM, a membrane permeable rapid Ca2+ chelater, completely inhibited the p38 phosphorylation (Fig. 4a3). The ATP-evoked p38 activation was largely dependent upon Ca2+, especially upon extracellular Ca2+. ATP (1000 µm) produced a transient increase in [Ca2+]i, which was followed by a sustained rise in [Ca2+]i(Fig. 4a). The transient component was little affected by the removal of extracellular Ca2+ (– Ca2+Fig. 4a1), but was inhibited by U73122 (1 µm), a selective PLC inhibitor (27.4 ± 10.9% of ATP alone, n = 21), and the sustained one was abolished by – Ca2+(Fig. 4a1). Thus, the transient and sustained components appeared to be due to Ca2+ release by inositol 1,4,5-trisphosphate (InsP3) and Ca2+ entry from the extracellular space such as store-operated Ca2+ entry (SOC), a Ca2+ entry dependent upon the filling state of the stored Ca2+ concentration (Berridge 1995), respectively. In addition to SOC, the sustained Ca2+ response to ATP would involve Ca2+ entry mediated by P2X7 receptors. In fact, BzATP (1000 µm) produced a sustained Ca2+ entry (Fig. 4b1). BzATP also evoked an extracellular Ca2+ dependent p38 phosphorylation that lasted over 10 min (Fig. 4b2). Although BzATP at 1000 µm stimulated the maximal IL-6 production, the BzATP (1000 µm)-evoked IL-6 production was less than 10% of that evoked by 1000 µm ATP (2.4% of ATP-evoked response) (Fig. 4b3i). The cells were not damaged by BzATP because almost all cells were stained with FAD 24 h after the treatment with BzATP (1000 µm). In addition, Brilliant Blue G (BBG), a selective P2X7 receptor antagonist (Jiang et al. 2000), did not inhibit the ATP-evoked IL-6 mRNA production (Fig. 4b3ii) in spite of the fact that BBG inhibited the BzATP-evoked elevation in [Ca2+]i by about 70% (34.2 ± 1.2% of BzATP alone, n = 7). Thus, although stimulation of P2X7 receptors activated p38 in an extracellular Ca2+ dependent manner, its phosphorylation was not sufficient to produce IL-6. It is suggested that a pathway(s) mediated by P2X7 receptors would not be dominant for the IL-6 production. The findings that the ATP-evoked IL-6 production was abolished by either N-ethylmaleimide (NEM) 10 µm, an inhibitor of some G-proteins (Rhee et al. 2000), or U73122 (1 µm) would also support the idea (below detectable level, 2 separate experiments). On the other hand, the phosphorylation by ATP of ERK1/2 was insensitive to extracellular Ca2+, but was abolished by BAPTA-AM (data not shown).

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Figure 4. Changes in [Ca2+]i and Ca2+ dependency of the phosphorylation of p38 evoked by ATP and BzATP. (a1) ATP-evoked increase in [Ca2+]i in MG-5. ATP (1 mm) was applied to the cells in the presence (solid line) and absence (dotted line) of extracellular Ca2+ as indicated by a solid bar. Data represent the mean of the responses in 10–11 cells from the same culture dish. At least three such independent experiments were performed and similar results were obtained. Ca2+ dependency of phosphorylation of p38 by ATP in MG-5 was shown in (a2) and (a3). ATP (1 mm) was applied to the cells in the presence (+ Ca2+) and absence (– Ca2+) of extracellular Ca2+ for 1 or 10 min (a2). The phosphorylation by ATP of p38 was dramatically inhibited by the removal of extracellular Ca2+. (a3), cells were treated with BAPTA-AM (100 µm) for 30 min, and then stimulated with 1 mm ATP for 1 min. These are results from a typical experiment. At least three such independent experiments were performed and similar results were obtained. (b1), the time-course of elevation in [Ca2+]i evoked by BzATP (100 and 1000 µm) in the cells in the presence of extracellular Ca2+. Data represent the average responses of 16 cells obtained from a microscopic field. At least three such independent experiments were performed and similar results were obtained. (b2) The time-course of the phosphorylation of p38 evoked by BzATP in MG-5. Cells were treated with 1 mm BzATP for the times indicated in the presence of extracellular Ca2+. These are results from a typical experiment. At least three such independent experiments were performed and similar results were obtained. Horizontal and vertical scale bars show 10 s and fura-2 fluorescent ratio (F340/F360 = 0.5), respectively. (3i) After treatment of the cells with ATP (1 mm) and various concentration of BzATP (100–5000 µm) for 24 h, the supernatant in each well was collected, and released IL-6 was measured. Values show mean ± SEM of five independent experiments. (ii) Effect of briliant blue G (1 µm, + BBG) on the ATP-evoked IL-6 mRNA expression. The cells were treated with BBG for 10 min, and then stimulated with 1 mm ATP for 6 h. After treatment of the cells with ATP in the presence or absence of the antagonists for 6 h, IL-6 mRNA expression was measured by quantitative RT-PCR. Data were normalized by the control. Values show mean ± SEM of four independent experiments.

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Involvement of Ca2+ dependent PKCs in the ATP-evoked IL-6 production in MG-5

In addition to InsP3 production, the activation of PLC results in diacylglycerol generation, subsequently leading to the activation of PKC. We therefore examined the effect of a PKC inhibitor on the ATP-evoked IL-6 production. Gö6976 (10 µm), a preferential inhibitor of Ca2+ dependent isoforms of PKC, significantly suppressed the ATP-evoked IL-6 mRNA production (43.4 ± 6.7% of control, n = 3) without affecting the p38 activation (Fig. 5). A short-term stimulation (10 min) with phorbol-12-myristate-13-acetate (PMA; 100 nm) enhanced the IL-6 mRNA expression (125.0 ± 0.6% of ATP alone, n = 3). Thus, in addition to p38, Ca2+ dependent isoforms of PKC were also involved in the ATP-dependent induction of IL-6 expression.

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Figure 5. Effect of Gö6976, an inhibitor of Ca2+-dependent PKC isomer, on the ATP-evoked IL-6 mRNA expression. Cells were treated with Gö6976 (1 or 10 µm) 10 min before and during ATP (1 mm) stimulation. Six hours after the initial ATP stimulation, IL-6 mRNA expression was measured by quantitative RT-PCR. Similarly, effect of Gö6976 on the ATP-evoked p38 phosphorylation was investigated (inset). At least three such independent experiments were performed and similar results were obtained. Asterisk on the column shows significant difference from the response evoked by ATP alone (*p < 0.05).

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P2 receptors responsible for the ATP-evoked IL-6 production in MG-5

Finally, the subtypes of P2 receptors responsible for IL-6 expression were examined. We conducted RT-PCR analysis using cDNA coding for P2Y and P2X7 receptors, and found the expression of mRNAs encoding P2Y1, P2Y2, and P2Y6 mRNAs in MG-5, whereas P2Y4 mRNAs were not detected under these PCR conditions (Fig. 6). The effect of PTX on the ATP- and ADP-evoked responses was also investigated. The ATP-evoked rises in [Ca2+]i, and the phosphorylation of p38 and ERK1/2 were not affected by PTX (Fig. 7a1, b1), whereas the ADP-evoked elevations in [Ca2+]i and the phosphorylation of ERK1/2 were strongly inhibited by PTX (Fig. 7a2, b2). Both ATP and ADP seemed to cause these responses via distinct P2Y receptors. With regard to the Ca2+ responses, both the ATP- and ADP-evoked transient increases in [Ca2+]i showed similar, characteristic features, i.e. both were independent of extracellular Ca2+ but sensitive to U73122 (27.4 ± 10.9% of ATP alone, n = 21; 38.5 ± 4.5% of ADP alone, n = 32). Thus, it is likely that ATP- and ADP-sensitive P2Y receptors are linked to PLC but only the P2Y receptor for ADP is coupled to PTX-sensitive G-protein.

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Figure 6. Detection of mRNAs for metabotropic P2Y and ionotropic P2X7 receptors in MG-5. The expression of mRNAs encoding various P2Y and P2X7 receptors in MG-5 were analyzed by a RT-PCR method using P2Y1, P2Y2, P2Y4, P2Y6 and P2X7 specific primers shown in Materials and methods.

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image

Figure 7. Effects of PTX on the ATP-evoked increase in [Ca2+]i (a) and p38 and ERK1/2 activity (b) in MG-5 cells. (a) The time-course of changes in [Ca2+]i evoked by 1 mm ATP (a1) and 1 mm ADP (a2) in PTX-treated (PTX; dashed traces) and -untreated (bold traces) cells. Cells were incubated with PTX 100 (ng/mL) overnight at 37°C and then stimulated with ATP or ADP. Each trace represents the average of the responses obtained from 10 to 16 cells. At least three such independent experiments were performed and similar results were obtained. (b1) PTX had no effects on the ATP-evoked phosphorylation of both p38 and ERK1/2, but almost abolished the ADP-evoked phosphorylation of ERK1/2 MAP and slightly inhibited the p38 (b2). Cells were treated with 100 ng/mL PTX, and then stimulated with 1 mm ATP for 1 min (p38) and 30 min (ERK1/2) or with 1 mm ADP for 1 min (p38 and ERK1/2). These are results from a typical experiment. At least three such independent experiments were performed and similar results were obtained. Asterisks show activated forms of MAP kinases.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In the present study, we demonstrated that extracellular ATP, acting on PLC-linked P2Y receptors, stimulated the expression of IL-6 mRNA and the subsequent production of IL-6 via the activation of p38 and Ca2+ dependent PKC in MG-5. This is a novel pathway for the induction of cytokines in microglia.

ATP is easily metabolized into ADP, AMP and adenosine by ecto-ATPase and 5′-nucleotidase, but these metabolites were not involved in the IL-6 induction for the following reasons: (1) neither ADP nor adenosine stimulated the IL-6 production (2) aminophylline had no effect on the ATP-evoked IL-6 release. In addition, suramin (1 mm) abolished the ATP (1 mm)-evoked IL-6 mRNA expression almost completely. Thus, the ATP-evoked IL-6 production was due to the activation of P2 receptors in MG-5.

ATP might evoke the release of IL-6 secondarily by releasing other cytokines such as TNF-α and IL-1β. In fact, TNF-α and IL-1β could stimulate IL-6 production in several glial cells (Norris et al. 1994). In our present experiments, when MG-5 were treated with 1000 µm ATP, a significant amount of TNF-α appeared at 1 h and peaked at 3 h after the stimulation (data not shown). The released TNF-α might stimulate the release of IL-6, leading to the slow time-course of IL-6 release. TNF-α (murine rTNF-α), however, never produced the release of IL-6 in MG-5 even when its concentration was raised to 10 000 pg/mL (data not shown). This result was well in accordance with a previous report that TNF-α failed to stimulate IL-6 production in microglia (Sawada et al. 1992). Moreover, the release of IL-1β, a potent inducer of IL-6 gene (Lee et al. 1993), was not evoked by 1000 µm ATP (data not shown). It is also possible that other mediators generated secondarily by ATP might be a trigger for the signaling cascade and IL-6 production. However, when the cells were incubated with ATP for shorter periods (60 min), and even when they were washed out thoroughly, the IL-6 mRNA induction was still observed [about 70% of ATP (6 h) evoked response]. Thus, although we can not completely exclude the possibility that other mediators generated secondarily by ATP might also be involved in this signaling cascade, it is suggested that the activation of P2 receptors by ATP and their intracellular signals would be directly involved in the IL-6 mRNA expression and IL-6 release.

We showed that ATP could activate two distinct MAP kinases, i.e. ERK1/2 and p38, in MG-5. Also, ATPγS activated both MAP kinases. ADP strongly activated ERK1/2 but only slightly phosphorylated p38 (Fig. 2c). Theses results, when considered together with those concerning the IL-6 release, strongly suggest that p38 but not ERK1/2 MAP kinase is responsible for the IL-6 production. This hypothesis was proved by the experiment using selective inhibitors of p38 and ERK1/2 (Fig. 3). It should be noted that, although ATP activated two MAP kinases, p38 was the one responsible for the IL-6 gene expression in the cells. Thus, each MAP kinase would be linked to distinct physiological functions in the cells. In contrast, Hide et al. (2000) have reported that ATP triggers TNF-α release by a mechanism that is dependent on both the ERK1/2 and p38 cascades in rat microglia. In MG-5, the release of TNF-α evoked by ATP was also abolished by both ERK1/2 and p38 inhibitors (data not shown). Each cytokine seems to be regulated by its own intracellular signaling pathway(s).

Previous studies with IL-1β−stimulated synoviocytes indicated that p38 activation leads to IL-6 gene expression through an enhancement of IL-6 mRNA stability (Miyazawa et al. 1998). However, in our experiment, p38 appeared to act on the initial ATP signal transduction pathway affecting IL-6 gene expression, as no significant decrease of IL-6 mRNA expression was observed when SB203580 was added 3 or 5 h after the ATP application. The finding that the ATP-evoked phosphorylation of p38 was rapid, i.e. peaking at 1 min and almost disappearing at 60 min after ATP stimulation (Fig. 2a), may also exclude the possibility that p38 activation leads to increased IL-6 expression via its effects on IL-6 mRNA stability.

The phosphorylation by ATP of p38 was dependent on the extracellular Ca2+. ATP produced a PLC-dependent transient Ca2+ release by InsP3, which was followed by sustained Ca2+ entry via both SOC and P2X7 receptors. Several groups have reported that P2X7 receptors have a central role in the production of cytokines in microglia (Ferrari et al. 1997a; Hide et al. 2000). In fact, BzATP evoked sustained Ca2+ entry via P2X7 receptors, leading to the phosphorylation of p38 in MG-5. However, BzATP induced only a small amount of IL-6 production in the cells (Fig. b3i). It is likely that, in contrast to previous reports in microglial cells, activation of P2X7 receptors is not necessarily required for IL-6 production in the cells. Instead, P2Y receptors appear to have a crucial role in the IL-6 induction. The most recent observation that ATP may work through P2 receptors other than P2X7 receptors to affect IL-6 production in LPS-primed P2X7-deficient mice (P2X7R–/–; Solle et al. 2001) may support our hypothesis. However, a requirement of the high ATP concentrations for the IL-6 production may still support the idea that P2X7 rather than P2Y receptors might be involved in the responses. Very recently, it has been reported that ATP acting on P2Y receptors stimulated the release of IL-12 in human dendritic cells (Wilkin et al. 2001). The ATP concentrations required for this IL-12 induction were similar to those required for the IL-6 production in MG-5. Thus, there seems to be a big variety in the sensitivity to ATP among the subclass of P2Y receptors. The deduced P2Y receptors in MG-5 therefore would be ones that require the high ATP concentrations for their activation. In addition, when cells were incubated with either NEM, an inhibitor of some G-proteins (Rhee et al. 2000), or U73122, the ATP-evoked IL-6 production was abolished (below detectable level). Thus, activation of PLC-linked P2Y receptors, rather than P2X7 receptors, and their receptor-mediated signals including p38 would trigger the IL-6 synthesis in MG-5. These results also suggest that phosphorylation of p38 is not sufficient to induce IL-6 release, and would predict the existence of additional signals for the IL-6 production.

ATP acts on PLC-linked P2Y receptors. The activation of PLC results in the formation of InsP3 and diacylglycerol, leading to the activation of Ca2+ release from InsP3-sensitive Ca2+ stores and PKC, respectively. We therefore examined the involvement of a PKC-mediated pathway(s) in the ATP-evoked IL-6 production and found that Gö6976, an inhibitor of Ca2+-dependent PKCs, inhibited the ATP-evoked IL-6 mRNA expression without affecting the p38 phosphorylation (Fig. 5). In addition, PMA (100 nm) up-regulated the IL-6 gene expression evoked by ATP or even BzATP (180.04% of BzATP alone). The simplest interpretation for the results would be that p38 and Ca2+-dependent PKC seem to work independently to control the IL-6 production. Thus, the ATP-evoked IL-6 production seems to be regulated by at least two independent pathways, p38 and Ca2+-dependent PKC. It is unknown what the relative contributions of these intracellular signals in causing IL-6 gene expression are or which downstream signals are involved in the IL-6 gene expression. Some transcriptional factors such as NF-κB p65 (RelA) (Ferrari et al. 1997b), Jun, and Fos (Neary et al. 1996b) are known to be activated by ATP. Berghe et al. (1998) have described the involvement of MAP kinase pathways in NF-κB transactivation, which leads to the induction of IL-6 gene expression. Although such transcriptional factors may work as the downstream signals of either p38 or Ca2+-dependent PKC, the detailed mechanism underlying such a cooperative regulation of IL-6 production remains to be clarified.

Finally, we tried to determine the subclass of P2Y receptors involved in the ATP-evoked responses. RT-PCR analysis showed that P2Y1, P2Y2 and P2Y6 but not P2Y4 mRNAs are present in the cells (Fig. 6). However, the pharmacological profile for recombinant P2Y1, P2Y2, and P2Y6 receptors is inconsistent with our findings on the IL-6 gene expression by P2 agonists, as neither ADP, a potent agonist to P2Y1, nor UTP, an activator of P2Y2 and P2Y6 receptors, stimulated significant IL-6 gene expression in MG-5. This finding suggests that the properties of the P2Y receptors coupled to IL-6 gene expression in MG-5 differ from those of the recombinant P2Y receptors or that MG-5 express novel ATP-preferring receptors coupled to IL-6 production. Both ATP and ADP produced a transient Ca2+ release via PLC-linked P2Y receptors. However, only the ADP-evoked Ca2+ release was sensitive to PTX, suggesting that the responses evoked by ATP and ADP were mediated by distinct P2Y receptors (Fig. 7). As for the PTX-sensitive P2Y receptors, P2Y12, a new P2Y receptor subclass which has just been cloned (Hollopeter et al. 2001), may be the most probable P2Y receptor for ADP because AR-C67085, an antagonist to P2Y12 (Jarvis et al. 2000), inhibited the ADP-evoked elevation in [Ca2+]i and ERK1/2 phosphorylation without affecting the ATP-induced p38 activation (Shigemoto-Mogami et al., unpublished data). Although the definitive classification of P2Y receptors for the ATP-evoked IL-6 production remains to be determined, it is suggested that ATP, acting on the PTX-insensitive P2Y receptors linked to PLC, stimulates the synthesis of IL-6 through the pathways involved in p38 and Ca2+-dependent PKC.

Taken together, we demonstrated novel signaling pathways for IL-6 production mediated by P2Y receptors in MG-5, i.e. the activation of p38 and Ca2+-dependent PKC via PLC-linked PTX-insensitive P2Y receptors. Although other functional P2 receptors are also present in the cells, their activation was not involved in the IL-6 production. ATP may regulate a variety of microglial functions distinctively via multiple P2 receptors in microglia. As MG-5 are a microglial cell line, they might not precisely reveal the same protein expression pattern and corresponding functional profile as microglia in situ. However there are several important similarities between MG-5 and microglia (Ohsawa et al. 1997). Thus, the present findings observed in MG-5 would be important as this novel signaling pathway for IL-6 production would be present and work in microglia either in physiological or pathophysiological conditions in situ.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank Dr J. Neary for reading and improving the manuscript and Dr K. Sato for helpful suggestions concerning the cell viability experiment. This study was supported partly by the promotion of fundamental studies in Health Science of the Organization for Pharmaceutical Safety and Research.

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  5. Discussion
  6. Acknowledgements
  7. References
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