Microglial cells express a peculiar plasma membrane receptor for extracellular ATP, named P2Z/P2X7 purinergic receptor, that triggers massive transmembrane ion fluxes and a reversible permeabilization of the plasma membrane to hydrophylic molecules of up to 900 dalton molecule weight and eventual cell death (Di Virgilio, F. 1995. Immunol. Today. 16:524–528). The physiological role of this newly cloned (Surprenant, A., F. Rassendren, E. Kawashima, R.A. North and G. Buell. 1996. Science (Wash. DC). 272:735–737) cytolytic receptor is unknown. In vitro and in vivo activation of the macrophage and microglial cell P2Z/P2X7 receptor by exogenous ATP causes a large and rapid release of mature IL-1β. In the present report we investigated the role of microglial P2Z/P2X7 receptor in IL-1β release triggered by LPS. Our data suggest that LPS-dependent IL-1β release involves activation of this purinergic receptor as it is inhibited by the selective P2Z/P2X7 blocker oxidized ATP and modulated by ATP-hydrolyzing enzymes such as apyrase or hexokinase. Furthermore, microglial cells release ATP when stimulated with LPS. LPS-dependent release of ATP is also observed in monocyte-derived human macrophages. It is suggested that bacterial endotoxin activates an autocrine/paracrine loop that drives ATP-dependent IL-1β secretion.
With the recent cloning of the macrophage P2Z/ P2X7 receptor, purinergic receptors in immune cells are attracting increasing interest (1, 2). Microglial cells are a good model to investigate physiological functions of purinergic receptors in the immune system because neurons are among the few cell types that have been unequivocally shown to release ATP (3). A recent study from our laboratory has shown that exogenous ATP causes a large release of IL-1β from N9 and N13 microglial cell lines and freshly isolated microglial cells by activating the P2Z/P2X7 receptor (2). IL-1β has a pivotal role in several chronic inflammatory diseases and in the pathogenesis of septic shock (4–6). It is synthesized by activated macrophages and microglial cells as an inactive 33-kD precursor (proIL-1β) that is cleaved into the active 17-kD form by a cysteine protease named IL-1β–converting enzyme (ICE) (7, 8). Mature IL-1β is released through an as yet unknown pathway. In macrophages and microglial cells, bacterial endotoxin (LPS), the best characterized stimulant of IL-1β release, causes a rapid and large in vitro accumulation of proIL-1β, followed by slow release of the mature form (9). Release of processed IL-1β can be greatly accelerated by depletion of endogenous K+ (4, 10). This very recent observation has led to the hypothesis that in vivo an additional stimulus that causes cytoplasmic K+ depletion must act in synergy with LPS to induce fast IL-1β release (11). Extracellular ATP (ATPe) is an interesting candidate to serve this role because it causes massive K+ depletion via activation of the macrophage P2Z/P2X7 receptor (12). The present report suggests that the P2Z/P2X7 receptor is also involved in IL-1β release stimulated by LPS, presumably through an autocrine loop based on LPS-triggered ATP secretion.
Materials And Methods
Cell Culture and Cytokine Measurement.
N9 and N13 microglial cell lines were a kind gift of Dr. Paola Ricciardi-Castagnoli (University of Milano, Italy) and were grown in RPMI 1640 medium (PAA, Linz, Austria) supplemented with 2 mM glutamine and 10% (heat-inactivated) FCS (Life Technologies Ltd., Paisley, Scotland), 100 U/ml penicillin, and 100 μg/ml streptomycin as described previously (2). Human monocytes were isolated from buffy coats by one-step gradient (Percoll; Pharmacia Biotech SpA, Cologno Monzese, Italy) or by adherence on plastic Petri dishes. After isolation, cells were kept in culture for 5 d in RPMI medium containing 2 mM glutamine, 5% human serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. IL-1β and IL-6 in the supernatant of LPS (Sigma Chemical Co., St. Louis, MO) treated cells were measured with the Intertext-1βX mouse IL-1β ELISA kit and Intertext-6X mouse IL-6 ELISA kit, respectively (Genzyme srl, Cinisello Balsamo, Italy). All reagents used were dissolved in endotoxin-free water (Sigma) and checked for endotoxin contamination. Periodate-oxidised ATP (oATP) was synthesized by Dr. S. Hanau as described in reference 13.
Measurement of Extracellular ATP.
Microglial cells (25 × 103/ well) were plated in microtiter plastic wells in culture medium and incubated in a CO2 incubator at 37°C in the absence or presence of LPS for 24 h. At the end of this incubation, the monolayers were thoroughly rinsed with saline solution and supplemented with 100 μl of a special diluent buffer (FireZyme Ltd., San Diego, CA) to stabilize extracellular ATP and directly placed in the test chamber of a luminometer (FireZyme). Then, 100 μl of luciferin-luciferase solution (FireZyme) was added, and light emission was recorded. As a control, total cellular ATP content was also routinely monitored. Under resting conditions, extracellular ATP amounted to ∼10–15% of total.
Results And Discussion
Fig. 1 shows that a 24-h incubation in the presence of 10 μg/ml LPS triggers release of IL-1β and that this is blocked by pretreatment with the selective P2Z/P2X7 inhibitor (13) oATP. To show that the effect of oATP is not due to a nonspecific inhibition of cell responses, we have also monitored IL-6 release, which is much less affected. As further proof that oATP does not have nonspecific effects, we show that IL-1β release is restored in LPS-treated, oATPinhibited cells by the K+ ionophore nigericin, an agent known to cause IL-1β release through a receptor-independent pathway (4, 10).
Autocrine/paracrine stimulation of purinergic receptors can also in principle be prevented by exogenously added ATP-consuming enzymes such as apyrase or hexokinase. Fig. 2,A shows that apyrase completely inhibits LPS-dependent IL-1β release (the inactivated enzyme has no such effect). Surprisingly, hexokinase does not inhibit but rather stimulates IL-1β release. The main difference between apyrase and hexokinase is that the first hydrolyzes ATP and ADP, thus generating AMP, whereas hexokinase uses ATP as phosphorus donor to phosphorylate glucose, thus generating glucose 6 phosphate and ADP. It is known that ADP is an agonist at P2Z/P2X7 receptor, though less potent than ATP (12). Thus we checked whether the potentiating effect of hexokinase is mediated by stimulation of the P2Z/ P2X7 receptor by accumulated ADP. This seems to be the case because pretreatment with oATP blocks IL-1β secretion due to the combined addition of LPS and hexokinase (Fig. 2,A), and more importantly, exogenous ADP (ADPe) is a much more potent stimulus than ATP (Fig. 2 B). These experiments suggest that IL-1β release could be modulated by ATPe and ADPe, probably released by the inflammatory cells themselves under LPS stimulation.
An obvious sine qua non of this hypothesis is that microglial cells must release ATP in response to LPS. Fig. 3,A shows that microglial cells chronically stimulated with LPS release ATP. Because the incubation medium is changed right before ATP determination, extracellular ATP measured in this experiment is very likely not accumulated in the bulk phase but continuously generated by the microglial cells. In support of this interpretation, we consistently found very little extracellular ATP in the cell-free supernatant (not shown). This observation is consistent with that previously reported by Filippini et al. (14) in T lymphocytes. The LPS dose– response curve for ATP release closely matched that for IL-1β release, as shown in Fig. 3,B. It has been shown previously that ATP is a powerful stimulus for IL-1β secretion from macrophages (10, 11), thus suggesting that this nucleotide might also have a role in autocrine/paracrine stimulation of these cells. In support of this hypothesis, Fig. 4 shows that ATP is released by human macrophages isolated from three different subjects after stimulation with LPS.
The mechanism of IL-1β processing and release is a key issue in immunology (5–9, 15). Rather surprisingly, recent evidence points to a decrease in cytoplasmic K+ as a pivotal stimulus for ICE activation and IL-1β maturation (4, 10). However, LPS itself does not directly activate plasma membrane K+ channels, and mouse microglial cells express inwardly but not outwardly rectifying K+ channels (16), thus raising the issue of the mechanism responsible for lowering the cytoplasmic K+ concentration. It has been suggested that this might be achieved by a LPS-dependent increase in the number of voltage-dependent K+ channels in the macrophage plasma membrane (4), but typical K+ channel inhibitors blocked IL-1β release only at concentrations far above those necessary to inhibit these channels (4). The P2Z/P2X7 receptor is a good candidate to mediate cytoplasmic K+ depletion. This receptor is typically expressed in macrophages and macrophage-like cells (2, 17, 18), and it is modulated by inflammatory cytokines (17, 19). A brief stimulation with ATPe triggers massive K+ efflux (12) and release of processed IL-1β (2, 10, 11), whereas a sustained activation causes cell death (1, 17, 20). Our data suggest that IL-1β release from microglial cells requires a double stimulation: first, LPS-dependent transcription of the IL-1β gene and cytoplasmic accumulation of proIL-1β; second, paracrine/autocrine activation of the P2Z/P2X7 receptor that causes release of the mature cytokine. Adenine nucleotides can originate from many different sources: (a) the microglial cells themselves can release ATPe, either spontaneously or under LPS stimulation; (b) injured or damaged cells certainly release significant amounts of this nucleotide, a process likely to occur in vivo at sites of inflammation; (c) in the central nervous system, ATPe can be released by neurons that establish close contact with the microglial cells.
It might seem paradoxical that ADP is a better IL-1β– releasing agent than ATP, although notoriously it is a less potent stimulus at the P2Z/P2X7 receptor. However, this is not unexpected because ADP, in contrast with ATP, is devoid of cytotoxic activity, and data from our laboratory show that release of IL-1β is optimal in response to a submaximal, noncytotoxic stimulation of the P2Z/P2X7 receptor, such as that due to ADPe (D. Ferrari and F. Di Virgilio, manuscript in preparation).
Involvement of the P2Z/P2X7 purinergic receptor in LPS-dependent IL-1β release may allow the development of new pharmacological antagonists (i.e., oATP and derivatives) to modulate the in vivo production of this cytokine in pathological conditions such as septic shock or chronic inflammatory diseases.
We are grateful to Dr. Paola Ricciardi-Castagnoli (National Research Council and University of Milano) for the gift of N9 and N13 mouse microglial cell lines.
This work was supported by grants from the National Research Council of Italy (target project Clinical Application of Cancer Research, ACRO), the Ministry of Scientific Research (MURST), the Italian Association for Cancer Research (AIRC), the VIII AIDS Project, the I Tuberculosis Project, and Telethon of Italy.
Address correspondence to Francesco Di Virgilio, Institute of General Pathology, University of Ferrara, Via Borsari, 46, I-44100 Ferrara, Italy.