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- Materials and methods
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.
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.
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.
- Top of page
- Materials and methods
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.