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Department of Experimental and Diagnostic Medicine, Section of General Pathology, and Center of Biotechnology, University of Ferrara, Ferrara, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1ß. Both IL-1 and IL-1ß are synthesized
as 31–34-kDa procytokines and are then converted into the mature
17-kDa form; however, although IL-1 is biologically active in both
the pro and mature form, IL-1ß is only active in the mature form
(4). Conversion of pro into mature IL-1ß is catalyzed by
a cysteine protease known as IL-1ß-converting enzyme
(ICE)3
(3) that is the prototypical member of the caspase family
(caspase 1) (5, 6). Proteolytical maturation is also
linked via a poorly known mechanism to IL-1ß release into the
extracellular space. LPS is an efficient stimulus for IL-1ß release
from circulating monocytes, but when these cells are cultured in vitro
for a few days, they become refractory to LPS stimulation (7, 8). Likewise, peritoneal macrophages, microglial cells, and
dendritic cells respond weakly to LPS stimulation, thus suggesting that
a second stimulus may be needed to elicit IL-1ß secretion from these
cell types. Among stimuli reported to enhance and accelerate IL-1ß release from mononuclear phagocytes (cytolytic T cells, K+ ionophores, bacterial exotoxins, and ATP) (7, 8, 9, 10), ATP is one of the most interesting because this nucleotide is present at a concentration of 5–10 mM in the cytosol of most cells, thus it can be released in large amounts following plasma membrane damage or acute cell death. Furthermore, it is becoming increasingly clear that ATP is released by several cell types via nonlytic pathways in response to stimulation with many different agonists, among which is LPS itself (11, 12, 13, 14, 15). Once in the pericellular environment, ATP acts at plasma membrane P2 purinergic receptors, thus leading to cell activation. Mononuclear phagocytes express to high level a member of the P2X subfamily named P2X7 (also previously known as P2Z) that, upon ligation by ATP, mediates large transmembrane cation fluxes and, under extreme conditions of activation, the formation of nonselective plasma membrane pores (16, 17, 18). As reported by several laboratories, ATP, via activation of P2X7, is one of the most powerful stimuli for secretion of IL-1ß in its mature form (10, 19, 20, 21, 22). Rather interestingly, ATP is an efficient stimulus for IL-1ß secretion only after the cells have undergone a short priming with endotoxin, suggesting that this nucleotide is unable to trigger transcription of the IL-1ß gene, but rather acts posttranslationally, probably accelerating the step(s) involved in the proteolytical maturation of this cytokine.
It was originally proposed by us and others that ATP-mediated IL-1ß processing in macrophages and microglia is due to activation of ICE or a related caspase (8, 20, 23), but direct proof has not yet been provided. In the present work, we have investigated the mechanism of ATP-dependent IL-1ß release in mouse microglia. Our findings suggest that extracellular ATP, acting at the P2X7 receptor, triggers ICE activation and provides evidence for a crucial role of changes in the cytoplasmic K+ concentration as the coupling factor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Microglial N13 cells were obtained as described by Righi et al. (24) and cultured in RPMI 1640 medium supplemented with (Sigma, St. Louis, MO) 2 mM glutamine, 10% heat-inactivated FCS (Life Technologies Ltd., Paisley, Scotland), 100 U/ml penicillin, and 100 µg/ml streptomycin. Experiments were conducted in this culture medium without FCS. LPS (Sigma) was always used at a concentration of 1 µg/ml.
Measurement of enzymatic activity and IL-1ß release
Lactic dehydrogenase activity was measured as described previously (25). Intracellular and extracellular IL-1ß were measured with the same Endogen mouse IL-1ß ELISA kit (Endogen, Woburn, MA) that does not discriminate between the pro and mature cytokine forms. The caspase 1/ICE inhibitor YVAD-CHO and the caspase 3/apopain inhibitor DVAD-CHO were purchased from Bachem Feinchemikalen AG (Bubeudorf, Switzerland).
Immunoblotting and caspase 1/ICE activation
For IL-1ß detection, cells were lysed with 0.1% Triton X-100 solution in PBS. Cell lysates and supernatants were run on a 12% polyacrylamide gel (Merck, Milan, Italy) and blotted onto a reinforced nitrocellulose filter (Amersham International, Amersham, U.K.). IL-1ß was detected with a goat anti-mouse IL-1ß polyclonal Ab (Genzyme, Cinisello Balsamo, Italy) followed by staining with protein A labeled with HRP and visualization by chemiluminescence (Amersham). For caspase 1 detection, cell extracts were prepared by resuspending PBS-washed cell pellets in a high-salt buffer solution containing 20 mM HEPES (pH 7.9), 350 mM NaCl, 20% glycerol, 1% IGEPAL CA-630 (Sigma), 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 0.5 mM DTT, 2 mM PMSF, and 2 µg/ml aprotinin. Extracts were incubated on ice for 20 min and then cleared by centrifugation. Supernatants were run on a 15% polyacrylamide gel. The p20 caspase 1 proteolytic fragment was detected with peroxidase-conjugated affinity-purified goat anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA). Caspase 1/ICE activity was also measured with a fluorometric assay based on the cleavage of the YVAD-7-amino-4-trifluoromethyl coumarin (YVAD-AFC) specific caspase 1 substrate (Medical Biological Laboratories, Nagoya, Japan).
Data presentation
Data shown are averages ± SD of determinations from three to four experiments performed in duplicate. Sometimes error bars are not shown because their width exceeded that of the symbol.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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reports an experiment in which N13
cells were stimulated with LPS (filled circles) and the supernatants
withdrawn for IL-1ß determination at the indicated time points. In a
parallel experiment, the cultures were briefly pulsed for 30 min with
ATP (open circles) after the increasing intervals of LPS treatment. The
supernatants were then withdrawn for IL-1ß measurement. Addition of
ATP after a 2-h pretreatment with LPS increased accumulation of
extracellular IL-1ß from about 20 to 5000
pg/106 cells. Even more impressive was the
enhancement (from 100 to 100,000 pg/106 cells)
observed when ATP was added 6 h after LPS. The ability of ATP to
stimulate IL-1ß release decreased when the interval after LPS
addition was extended over 6 h, and after 24 h any
stimulatory activity was completely lost.
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A, lane 6).
ATP, on the contrary, promoted efficient cleavage of the cytokine when
added to cells that had been preincubated in the presence of LPS (Fig. 2A, lanes 3 and 7), although the
amount of the 17-kDa form was much larger in the supernatants of
microglial cells treated with LPS for 6 h (Fig. 2A,
lane 7) compared with those treated for 2 h
(lane 3). A short 5-min incubation with the
ICE/caspase 1 inhibitor YVAD before the addition of ATP completely
inhibited the appearance of the 17-kDa band (lanes 4
and 8) and also greatly decreased the release of the 34-kDa
band and other proteolytic fragments. The inhibitory effect of YVAD was
more marked when cells were primed with LPS for 2 h compared with
a 6-h priming (cf lanes 4 and 8). LPS by itself,
applied for 2 or 6 h, triggered no IL-1ß release whether in the
34- or 17-kDa forms (lanes 2 and 6).
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B) showed
a plateau at a concentration of about 3 mM, in agreement with previous
findings in mouse microglia (20), but slightly higher than
previously reported by our laboratory for human macrophages
(21).
We then measured the kinetics of accumulation of intracellular IL-1ß
in cells treated with LPS or LPS plus ATP (Fig. 3). LPS caused a maximal increase in the
intracellular IL-1ß content 6 h after stimulation and then
cytokine content declined to reach nearly undetectable levels after
24 h. Stimulation with ATP for 30 min slightly but consistently
enhanced the amount of intracellular IL-1ß, whether added 2 or 6
h after LPS, but this small increase was not statistically significant.
A comparison between Figs. 1 and 3 suggests that there is a close
correlation between the amount of the intracellularly available IL-1ß
and the amount that can be released by ATP, since peak release by this
nucleotide was achieved 6 h after pretreatment with LPS, when
availability of intracellular IL-1ß was maximal and the lowest
ATP-mediated release occurred after 24 h of LPS incubation, when
intracellular IL-1ß was almost undetectable. We would expect to find
a decrease of intracellular IL-1ß in the ATP-treated cells, as a
consequence of the large secretion triggered by this nucleotide, but to
our surprise we found no change or, at the most, a small increase, as
if ATP-stimulated cells not only released more IL-1ß, but also
accumulated more procytokine, secretion being probably the
rate-limiting step for further procytokine synthesis. If this were the
case, i.e., if ATP-dependent IL-1ß release depended in part on the
continuos neosynthesis of pro-IL-1ß, then it should be at least in
part sensitive to inhibition by blockers of protein synthesis. This is
indeed the case as addition of cycloheximide before ATP reduced
intracellular and extracellular IL-1ß accumulation by about 15 and
30%, respectively, compared with samples treated with ATP in the
absence of the inhibitor (Table I).
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showing that the ICE/caspase
1-selective blocker YVAD inhibited IL-1ß release with an
IC50 of about 1 µM, whereas the less selective
tetrapeptide DEVD was much less active (IC50, 10
µM), and even at high concentrations was unable to fully prevent
IL-1ß release. To further support a selective effect of YVAD on
ICE/caspase 1 processing, Fig. 5 shows
that this tetrapeptide had no effect on the accumulation of
pro-IL-1ß.
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suggests that this is also likely the
case in microglia since it shows that increasing the external KCl
content of a normal RPMI medium strongly inhibited ATP-dependent
IL-1ß release, although under these ionic conditions
P2X7 receptor activation was not inhibited (data
not shown). The reduced cytokine release was not due to hypertonicity
because the addition of NaCl had no or very little effect. Further
proof of the ability of ATP to turn on ICE/caspase 1 comes from the
detection of the p20 ICE/caspase 1 fragment in ATP-challenged cells
(Fig. 7). Although some p20 accumulation
was detectable in cells treated with ATP alone (lane
3), an unequivocal band was clearly detected only in the sample
that was treated with LPS before ATP addition (lane
2), suggesting that LPS treatment also increased synthesis of the
p45 ICE/caspase 1 precursor. Treatment with LPS in the absence of ATP
caused no p20 fragment formation (data not shown). We confirmed
activation of ICE/caspase 1 by ATP by measuring the cleavage of the
fluorogenic substrate YVAD-7-amino-4-trifluoromethyl coumarin
(YVAD-AFC) (Fig. 8). Cell lysates of
control or ATP-treated samples were incubated in the presence of the
fluorogenic substrate for increasing times (1–3 h), clearly showing
that although control samples (open circles) produced a negligible
increase in fluorescence, the ATP-treated samples (open triangles)
generated at least a 3-fold increase in fluorescence. It is of interest
that the mere treatment with LPS (open squares) caused no caspase 1/ICE
activation.
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compares the kinetics of
accumulation of intracellular and extracellular IL-1ß in the presence
of LPS (Fig. 9A) or LPS plus ATP (Fig. 9B). By
comparing intracellular and extracellular IL-1ß in microglia
stimulated with LPS alone, it can be observed that a very small
fraction of the total cellular cytokine was released (e.g., 6 h
after LPS, 50 pg/106 cells was found
extracellularly against about 20,000 pg/106 cells
found intracellularly); furthermore, after an initial phase during
which intracellular and extracellular IL-1ß accumulated in parallel,
intracellular IL-1ß declined while extracellular accumulation showed
a steady rate of increase. However, even at the point of maximal
release (24 h), extracellular IL-1ß was only a very small fraction of
the total, a finding that further supports the observation that LPS is
a very inefficient stimulus for secretion and that, in the absence of
release, most of the intracellular cytokine is degraded. In the samples
treated with LPS plus ATP, intracellular accumulation and release of
IL-1ß were more closely related (Fig. 9B). Maximal release
occurred when ATP was added 6 h after LPS. At later time points,
secretion showed a fast decline, presumably reflecting exhaustion of
the intracellular IL-1ß reservoir.
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Discussion |
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IL-1ß is synthesized as a 31–34-kDa procytokine that is converted into the mature 17-kDa biologically active form by a cysteine protease named ICE (1, 2, 3, 4), also known as caspase 1, this enzyme being the founder member of the caspase family. ICE/caspase 1 itself is synthesized as a 45-kDa precursor (p45) that is then proteolytically cleaved into the active (p10/p20)2 form (5). The enzyme(s) responsible for the cleavage of caspase 1 is unknown; however, since this cysteine protease is capable of autoprocessing, activation might be due to an autoproteolytic process.
It was initially reported by Perregaux and Gabel (10) and Walev et al. (26) that maneuvers aimed at reducing the intracellular K+ concentration, either by means of selective K+ ionophores or inhibitors of plasma membrane K+ channels, caused strong activation of IL-1ß release in its mature form. However, it was not until 1998 that formal demonstration was provided that reducing the K+ concentration led to ICE/caspase 1 cleavage (27). The activatory effect of low K+ could also be shown on the isolated recombinant enzyme. These observations raise an obvious question: assuming that the activatory pathway involving a reduction in intracellular K+ has a physiological relevance in ICE/caspase 1 activation, what is the natural agonist that may cause cellular K+ efflux, given that neither nigericin nor K+ channel blockers are expected to be physiologically present in the body? Extracellular ATP is clearly an interesting candidate to such a role.
It is now well ascertained that ATP functions as an extracellular messenger in the nervous system as well as in the gut, skin, cardiovascular apparatus, and immune system (28, 29, 30). This nucleotide can be released by different mechanisms such as secretory exocytosis, plasma membrane transporters, passive leakage across the plasma membrane, or massive efflux as a consequence of cell death (14, 31, 32, 33, 34). This latter mechanism might be very relevant for IL-1ß secretion since it is well known that cell damage is an important cause of inflammation and vice versa. Therefore, it is not unlikely that a significant concentration of extracellular ATP could build up at inflammatory sites. Besides passive leakage, some inflammatory cells are capable of actively releasing ATP in response to extracellular stimulation. Sitkovsky’ s laboratory (35) has shown that up to 15% of total cellular ATP can accumulate in the pericellular environment in response to stimulation of CTL with anti-CD3 or anti-TCR Abs. We and others have shown that macrophages and microglial cells secrete ATP when stimulated with LPS (14, 15, 36), not to mention platelets that store massive amounts of ATP within dense granules (37), and can therefore cause significant increases in the extracellular ATP concentration when activated. That inflammatory cells are physiologically exposed to extracellular ATP is also hinted to by the expression of ATP-hydrolyzing enzymes, such as ecto-diphosphohydrolase (ecto-apyrase) and ecto-ATPase, on the outer aspect of their plasma membrane (38, 39).
Altogether, controlled release of ATP, presence of specific ATP receptors, and powerful ATP-consuming systems points to the operation of an ATP-based autocrine/paracrine loop that might have an important role in the local modulation of inflammation (40). ATP has long been suspected to be involved in the short range modulation of inflammatory cells, but it has always been difficult to pinpoint a unique ATP-dependent response. Control of IL-1ß maturation and secretion is a good candidate function for extracellular ATP.
LPS, the best known stimulus for IL-1ß release from macrophages and microglial cells, is very inefficient in vitro because it causes little secretion and mostly in the 34-kDa unprocessed form. Thus, it is postulated that a second factor that triggers ICE/caspase 1 activation is needed. Although, on one hand, this two-step process reveals the existence of tightly controlled mechanisms for IL-1ß secretion, on the other it raises the question of the identity of the ICE/caspase 1-activating factor. Posttranslational IL-1ß processing has been achieved by treating LPS-primed mononuclear phagocytes with several factors: nigericin, cytolytic toxins, cytolytic T cells, and ATP (8, 9, 19, 20). Among them all, ATP is the only agent likely to have a widespread physiological role, since nigericin is an experimental tool, cytolytic toxins will only be present during bacterial sepsis, and, finally, in vivo generation of anti-macrophage cytolytic T cells is an unusual event. Thus, ATP could be the external trigger for the proteolytic cleavage and externalization of pro-IL-1ß accumulated intracellularly upon LPS stimulation. The data reported in the present work provide further support for the role of K+ in the coupling between P2X7 and ICE/caspase 1. ATP, via activation of P2X7, is well known to cause a large K+ efflux (10) and thus a drop in the cytoplasmic concentration of this cation. We wonder whether also the other biological agents capable of triggering IL-1ß maturation (e.g., bacterial toxins and cytolytic lymphocytes) might act through this same mechanism, since they also are membrane-perturbing agents. This interpretation is supported by the inhibitory effect on IL-1ß release observed by raising extracellular K+, a maneuver that prevents movement of this cation along its chemical gradient (outwardly directed), as shown earlier by Perregaux and Gabel (10). The study by Cheneval et al. (27) performed in a human monocyte cell line lends further support to this hypothesis by showing that enzymatic activity of recombinant ICE/caspase 1 also requires a drop in the K+ concentration. It is likely that in the intact cell, P2X7-dependent changes in cytoplasmic K+ will be quickly transmitted to ICE/caspase 1 since this enzyme is thought to be compartmentalized in the subplasmalemmal cytoplasm.
In the absence of ATP, the kinetics of IL-1ß accumulation and release from microglial cells closely matched that previously described in murine macrophages (7). Peak intracellular accumulation occurred 6 h after LPS addition, then a steady decline followed. Externalization was much slower, never reaching >3% of total (intracellular plus extracellular) IL-1ß, in agreement with previous data (7). In the presence of ATP, efficiency of the release process was dramatically enhanced. When added 2 h after LPS, ATP triggered the release of about 25% of the total cellular IL-1ß content (5,000 vs 20,000 pg/106 cells, respectively); however, when added 6 after LPS, ATP released >80% of total IL-1ß (125,000 vs 145,000 pg/106 cells, respectively). Acceleration of release was not due to an increased rate of cell death because, as previously shown (21, 22), for these short incubations ATP has no or minimal cytotoxic effect.
The ICE/caspase 1 inhibitor YVAD, and to a lesser extent DEVD, drastically inhibited ATP-dependent accumulation of extracellular but not intracellular IL-1ß, pointing to a tight coupling between maturation and release. On the contrary, it is of interest that in mouse macrophages the proteolytic process and release appear to be uncoupled since a large amount of unprocessed IL-1ß was detected in the supernatant of these cells challenged with ATP in the presence of YVAD (22). This observation might suggest that this ICE/caspase 1 blocker has a tissue-specific effect on IL-1ß secretion, and thus it might have a more potent anti-inflammatory effect in the brain than in other tissues.
In conclusion, our study shows that in mouse microglia IL-1ß secretion is a two-step process requiring first IL-1ß gene transcription and pro-IL-1ß accumulation, then activation of ICE/caspase 1 to promote maturation. The second stimulus consists of an unusual second messenger: a drop in the cytoplasmic K+ concentration. Among various physiological agents, extracellular ATP via the P2X7 receptor is the most likely candidate to the role of extracellular trigger for ICE/caspase 1 activation. The use of selective P2X7 antagonists as anti-inflammatory drugs might be worth exploring.
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Footnotes |
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2 Address correspondence and reprint requests to Institute of General Pathology, University of Ferrara, Via Borsari, 46, I-44100 Ferrara, Italy.
3 Abbreviation used in this paper: ICE, IL-1ß-converting enzyme.
Received for publication September 21, 1999. Accepted for publication February 15, 2000.
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and IL-1ß occurs via a similar mechanism. J. Immunol. 160:2469.
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M. Solle, J. Labasi, D. G. Perregaux, E. Stam, N. Petrushova, B. H. Koller, R. J. Griffiths, and C. A. Gabel Altered Cytokine Production in Mice Lacking P2X7 Receptors J. Biol. Chem., January 5, 2001; 276(1): 125 - 132. [Abstract] [Full Text] [PDF]
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V. B. Mehta, J. Hart, and M. D. Wewers ATP-stimulated Release of Interleukin (IL)-1beta and IL-18 Requires Priming by Lipopolysaccharide and Is Independent of Caspase-1 Cleavage J. Biol. Chem., February 2, 2001; 276(6): 3820 - 3826. [Abstract] [Full Text] [PDF]
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J. Lee, J. Hur, P. Lee, J. Y. Kim, N. Cho, S. Y. Kim, H. Kim, M.-S. Lee, and K. Suk Dual Role of Inflammatory Stimuli in Activation-induced Cell Death of Mouse Microglial Cells. INITIATION OF TWO SEPARATE APOPTOTIC PATHWAYS VIA INDUCTION OF INTERFERON REGULATORY FACTOR-1 AND CASPASE-11 J. Biol. Chem., August 24, 2001; 276(35): 32956 - 32965. [Abstract] [Full Text] [PDF]
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) to measure stimulation of IL-1ß release
triggered by P2X7 receptor activation.
, IL-1ß release from samples incubated
with ATP in the absence of added LPS.
) or LPS plus 5 mM ATP (30 min, 
). 