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*
Institute of Medical Microbiology and Hygiene and
Pharmacological Institute, Johannes Gutenberg University, Mainz, Germany
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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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The starting point of the present studies was the recognition that K+ depletion triggers IL-1ß maturation (4, 5). Two mutually nonexclusive mechanisms could be responsible for this effect. First, intracellular K+ may have an influence on activation of pro-caspase-1 or on the activity of the assembled enzyme. Indeed, Cheneval et al. (6) recently presented evidence that K+ depletion promoted processing and autoactivation of caspase-1. A second possibility is that K+ depletion might facilitate cleavage of pro-IL-1ß by the protease, and this possibility was addressed in the present study. We report that the activities of cellular phospholipase A2 (PLA2)3 play a central, hitherto unrecognized, role in the regulation of IL-1ß processing. Thus, K+ depletion results in enhanced activity of the Ca2+-independent PLA2 (iPLA2). In contrast, calcium influx which activates Ca2+-dependent PLA2 (cPLA2) is apparently associated with an inhibitory influence on IL-1ß processing. The results reveal a connection between the activity of iPLA2 and the process of IL-1ß maturation in human monocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bromoenol lactone (BEL) and arachidonyl
trifluoromethylketone (AACOCF3) were
obtained from Alexis (Gruenberg, Germany). All other chemicals were
purchased from Sigma (Deisenhofen, Germany). The phospholipids and the
fatty acids were dissolved in 30 mM KOH and gently heated when
required. Fresh preparations were used. The final concentration of KOH
in cell cultures was 
300 µM. KCl buffer contained 150 mM KCl, 10 mM
HEPES, 1 mM MgCl2, 1 mM
CaCl2, and 1 g/L D-glucose. The pH
was adjusted to 7.4. NaCl buffer contained 150 mM NaCl, 10 mM HEPES, 1
mM MgCl2, 1 mM CaCl2, and 1
g/L D-glucose (pH 7.4).
Preparation and activation of human monocytes
Human peripheral blood mononuclear cells were obtained from fresh blood donated by healthy volunteers. Monocytes were isolated as described previously (4, 7). The monocytes were resuspended at 1 x 106 cells in RPMI 1640 (Life Technologies, Eggenstein, Germany) supplemented with 10% autologous human serum, 1 mM L-glutamine, and 100 U/ml penicillin-streptomycin and plated in 24-well plates (Nunc, Wiesbaden, Germany). After incubation for 1 h at 37°C, nonadherent cells were removed and the remaining cells were activated with 50 ng/ml LPS (Escherichia coli serotype 026:BH6; Sigma) for 4 h.
Determination of cellular K+
Monolayers of cells cultured in 6-well plates were incubated in the appropriate medium for 60 min at 37°C. The cells were then washed twice with 150 mM NaCl with 20 mM HEPES (pH 7.4) and lysed with 0.5% Triton X-100. Determination of K+ concentrations in the cell lysates were performed in an atomic absorption spectrophotometer (type AA-5; Varian-Techtron, Melbourne, Australia).
Transient expression of pro-1ß and of IL-1ß in COS-7 cells
cDNAs encoding the precursor and the mature form of IL-1ß were obtained by RT and amplification from total RNA of THP-1 cells. The products were cloned into the expression vector pCR3 (Invitrogen, Groningen, The Netherlands). Sequences of individual clones were verified by automated sequencing and plasmid for transfection was obtained by two rounds of cesium chloride density gradient centrifugation. The TNT-coupled transcription translation system (Promega, Mannheim, Germany) was employed to check the size of the proteins expressed from these constructs. COS-7 cells were seeded into 24-well plates at a density of 75,000 cells/well 24 h before transfection. A total of 400 ng of the respective expression plasmid was transfected per well using a calcium phosphate precipitation procedure as described earlier (8). Forty-eight hours after transfection, cells were treated and subsequently assayed for cytokine release in the supernatants as described for monocytes. Cell viability was checked by the trypan blue dye exclusion test. It was ascertained that <1% of the cells were trypan blue-positive at the termination of experiments.
Measurement of PLA2 activity
To determine PLA2 activity, monocytes were stimulated with 50 ng/ml LPS and prelabeled with [3H]choline (1 µCi/ml, overnight; NEN Life Science, Cologne, Germany) in MEM supplemented with 2% FCS. The cells were washed three times with medium and treated with appropriate reagents or buffers at 37°C for 60 min. At the end of the incubation, cells were extracted with chloroform-methanol, and unlabeled GPC was added as internal standard. The metabolites of the hydrophilic phase were separated by TLC (Merck no. 10845; Merck, Darmstadt, Germany) using methanol-1.5% NaCl-NH3 (10:10:1) as eluent, stained by iodine vapor, and the spot corresponding to GPC (Rf = 0.61) was scraped off and counted for radioactivity (9).
Arachidonic acid (AA) release
Cells were labeled with 0.4 µCi/ml [3H]AA in medium containing 50 ng/ml LPS for 4 h. The cells were then washed three times and treated as indicated in the figure legends. Released radioactivity from the supernatants was quantified by liquid scintillation counting.
Quantification of IL-1ß, pro-IL-1ß, and TNF-
Quantification of IL-1ß, pro-IL-1ß, and TNF- was
undertaken following the instructions supplied by the manufacturer of
the ELISA kits. The IL-1ß ELISA and TNF- ELISA kits were supplied
by BioSource (Ratingen, Germany) and the pro-IL-1ß ELISA kit by
Cistron (Pine Brook, NJ). The caspase-1 assay kit was provided by
Biomol (Hamburg, Germany).
Western blot analysis
Monocytes were stimulated with 50 ng LPS for 4 h. Lysis
buffer (150 mM NaCl, 0.2 mg/ml PMSF, 2 µg/ml aprotinin, 2 µg/ml
leupeptin, 2% Nonidet P-40, and 1% sodium deoxycholate) was added
(1:1) to the medium for 30 min at 4°C. Labeling and
immunoprecipitation was performed with a commercially available kit
(cellular labeling and immunoprecipitation kit; Boehringer Mannheim,
Mannheim, Germany). Proteins in the cell lysates were biotinylated with
D-biotinoyl-
-aminocaproic acid
N-hydroxysuccinimide ester, reacted with anti-human
IL-1ß polyclonal Ab (Endogen, Woburn, MA), and immune complexes were
precipitated with protein A-Sepharose and separated on 13%
SDS-polyacrylamide gels. After electrophoresis, the gel was blotted
onto polyvinylidene difluoride membrane and the biotinylated proteins
were visualized using streptavidin-peroxidase and chemiluminescence (BM
chemiluminescence blotting substrate; Boehringer Mannheim).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The first series of experiments used monocytes that were
stimulated with 50 ng/ml LPS to induce the release of mature IL-1ß.
After 4 h, IL-1ß production was unaffected by the presence of
indomethacin, an inhibitor of cyclooxygenase, or phenidion, an
inhibitor of lipoxygenase (10). However, IL-1ß release
was abrogated by AACOCF3, an inhibitor of both
cPLA2 and iPLA2
(11) (Fig. 1
A).
To specifically suppress the activity of the latter phospholipase
iPLA2, we employed the specific inhibitor BEL
(12, 13). In the presence of this agent, release of
IL-1ß was also effectively suppressed (Fig. 1A). SDS-PAGE
revealed that the absence of immunoreactive IL-1ß correlated with the
absence of the 17-kDa polypeptide (Fig. 1B). At the same
time, the Western blot showed that BEL did not deplete the cells of
pro-IL-1ß; hence, suppression of IL-1ß release was due to
inhibition of pro-IL-1ß processing. As shown in Fig. 2, half maximal inhibition was observed
at 2 µM BEL, a concentration at which this agent is only known to
interfere with the activity of iPLA2
and, additionally, with cytosolic phosphatidic acid phosphohydrolase
(PAP) 1 which forms diacylglycerol (DAG) from phosphatidic acid
(11). To exclude a participation of PAP, we applied
1,2-dioctanoyl-sn-glycerol, a membrane-permeable DAG, and
propranolol, a PAP inhibitor (14). Both agents did not
significantly affect IL-1ß processing (data not shown).
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addition of BEL at any time point immediately caused cessation of
IL-1ß maturation.
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or impair export of
IL-1ß
To determine whether BEL interfered with LPS-triggering events or
with the IL-1ß export machinery, monocytes were stimulated with LPS
in the presence or absence of 20 µM BEL, and the secreted amounts of
TNF- and pro-IL-1ß were quantified. As shown in Fig. 4, there was no effect of BEL on
LPS-induced TNF- secretion or pro-IL-1ß export. Thus, BEL appeared
to interfere neither with the LPS-signaling cascade nor with the export
machinery for pro-IL-1ß.
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BEL does not inhibit caspase-1
The activity of isolated caspase-1 was assessed in the presence and absence of 100 µM BEL or 100 µM AACOCF3 with the use of the caspase-1 assay kit. It was found that the activity of the protease was not influenced by the presence of either PLA2 inhibitor (data not shown).
Stimulation of iPLA2 enzymatic activity induced by K+ efflux
Since we and others (4, 5) had found that depletion
of cellular K+ levels promoted IL-1ß maturation
and, on the other hand, IL-1ß processing appeared to be dependent on
augmentation of iPLA2 activity, we decided to
test whether K+ depletion might enhance the
enzymatic activity of iPLA2. For this purpose,
cells were stimulated for 4 h with LPS to initiate IL-1ß
production which was followed by three experimental protocols. First,
the K+ ionophore nigericin was applied to provoke
selective loss of K+. Second, the cells were
treated with the pore-forming staphylococcal toxin which
permeabilizes the plasma membrane for monovalent ions (4, 16). Third, cells were exposed to medium devoid of
K+; this led to a gradual loss of cellular
K+ in the absence of membrane perturbation (our
experiments and Refs. 17, 18). Mature IL-1ß was
quantified in cell supernatants after 60 min. In parallel,
PLA2 activity was assessed by quantification of
glycerophosphocholine (GPC), a specific product of
PLA2-induced phosphatidylcholine degradation
(19) in the cell homogenates. Experiments were conducted
in the presence or absence of 20 µM BEL.
As summarized in Table I, both nigericin
and toxin provoked an increase in GPC concomitant to IL-1ß
maturation. Similarly, immersion of cells in
K+-free medium (NaCl) was accompanied by
increases in GPC and by IL-1ß processing. GPC formation and IL-1ß
maturation were totally abrogated in the presence of BEL, which itself
had no effect on the K+-depleting action of
either agent. Furthermore, the presence of high extracellular
K+ inhibited the generation of GPC in parallel to
inhibiting IL-1ß maturation. These results demonstrate that
iPLA2 is indeed activated when cells lose
K+.
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Calcium influx inhibits IL-1ß maturation
We also used the protocol of the foregoing experiment to test the
effect of the calcium ionophore A23187. Rather surprisingly, we
observed a reduction in secreted IL-1ß (Fig. 5). To test whether activation of the
cPLA2 isoform can be linked to IL-1ß
processing, we exposed the
[3H]arachidonate-prelabeled LPS-stimulated
cells to A23187 for 20 min, transferred the cells to
K+-free medium, and quantified the release of
[3H]AA as a parameter for
cPLA2 activation. Control cells that were
depleted of K+ without A23187 pretreatment
secreted IL-1ß into the supernatants in the absence of
[3H]AA liberation (Fig. 5). Cells that were
treated with A23187 alone released enhanced quantities of
[3H]AA, reflecting activation of
cPLA2, but did not secrete IL-1ß. Notably, when
cells were first treated with the ionophore and then depleted of
K+, processing of mature IL-1ß was markedly
suppressed compared with controls. These results are compatible with
the idea that cPLA2 and
iPLA2 counterbalance each other with regard to
the effect on IL-1ß maturation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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toxin was also suppressed by BEL. 3) K+
depletion resulting from incubation of cells in
K+-free buffer caused IL-1ß maturation that was
inhibitable by BEL. A contribution of the only other enzyme known to be
sensitive to micromolar concentrations of BEL, PAP type 1
(11), was excluded by the finding that a
membrane-permeable DAG analogue, 1,2-dioctanoyl-sn-glycerol,
did not overcome the inhibitory effects of BEL.
When PLA2 activation occurring concomitant to
K+ efflux and IL-1ß processing was directly
assessed by quantification of GPC, the contribution of
iPLA2 to GPC formation was again indicated by the
inhibitory influence of BEL. We originally attempted to quantify the
formation of lyso-PC, the primary product of PLA2
enzymatic activity. However, the results of lyso-PC determinations were
variable, probably due to the rapid breakdown (reacylation or further
hydrolysis to GPC) of this intermediate. Assessing GPC offered the
advantage that GPC has a longer half-life in the cell than lyso-PC and
yields less variable data (9). GPC is a specific indicator
of PLA2 activity because enzymatic hydrolysis of
phosphatidylcholine by PLA2 is the only metabolic
pathway which leads to GPC formation (19). Our results
(Table I
) indicated that K+ depletion effected by
different treatments indeed enhanced GPC generation in a BEL-sensitive
manner. Thus, our data indicate that activation of
iPLA2 can be added to the short list of processes
that have been found to be influenced by cellular
K+ levels. These include the formation of coated
pits and receptor-mediated endocytosis (20), cell
polarization (17), the activity of the molecular chaperone
heat shock protein 70, which has a K+ binding
site (21), and FAS-induced apoptosis (22).
We ascertained that BEL did not interfere with pro-IL-1ß or IL-1ß
secretion in transfected COS cells; therefore, BEL-dependent
suppression of IL-1ß maturation was obviously not due to perturbation
of the export machinery. BEL also was shown not to directly inhibit
caspase-1. Faced with the fact that the PLA2
inhibitor totally suppressed IL-1ß maturation in LPS-stimulated
cells, we are led to consider the possibility that
iPLA2 activation may be important for targeting
the substrate to its cleaving enzyme. There is evidence to indicate
that pro-IL-1ß is produced in the cytosol but translocates into
lysosome-related vesicles, where it presumably finds its cleaving
enzyme caspase-1 (23). It also appears that mature
caspase-1 is cosecreted with IL-1ß to the extracellular medium
(24). On this premise, it is conceivable that
iPLA2 activity is required to facilitate passage
of the precursor cytokine to the enzyme. This requirement may be
dependent on the concentrations of pro-IL-1ß and caspase-1: when
their levels reach a threshold, basal iPLA2
activity is sufficient for IL-1ß maturation to occur. When levels are
low, enhanced iPLA2 activity is required, and
this in turn can be effected by lowering cellular
K+ concentrations. The differences in LPS
responses of monocytes vs macrophages with regard to IL-1ß release
may be related to this issue. In contrast to monocytes, LPS stimulation
of macrophages leads to no appreciable release of IL-1ß unless
K+ efflux is induced by nigericin or ATP
(5) or toxin (our unpublished data). An unproven
possibility is that the basal level of iPLA2 in
macrophages is lower than that in monocytes, and therefore it is
essential to enhance activity by K+ depletion. We
found that BEL was equally efficient in suppressing IL-1ß maturation
in permeabilized macrophages (data not shown).
Attempts to stimulate pro-IL-1ß maturation by application of palmitic acid and AA, two products of iPLA2 action, to the cells were unsuccessful. When lyso-PC (100 µM) was applied, immunoreactive IL-1ß did become detectable in the supernatants, but SDS-PAGE revealed that the released cytokine differed in m.w. from the bona fide caspase-1 product (data not shown). Hence, we could not conclude that lyso-PC is the metabolite that promotes IL-1ß maturation. Perhaps cleavage and loss of membrane phospholipids by iPLA2 directly causes local membrane remodeling, which in turn is required for translocation of pro-IL-1ß into the trafficking vesicles or for its efficient cleavage by caspase-1. Our inability to copy the effect of iPLA2 activation through application of a metabolite may, of course, also have been due to more trivial problems of accessibility. Thus, if the targets for the iPLA2 metabolite were located intracellularly, e.g., in lysosome-related vesicles, it may not be possible to mimic the effect by extracellular application.
Application of the Ca2+ ionophore A23187 led to the expected generation of AA, indicative of cPLA2 activation. Surprisingly, this was accompanied by reduction of IL-1ß maturation. The possibility is thus considered that iPLA2 and cPLA2 constitute a regulatory system with mutually counterbalancing roles in the regulation of caspase-1 function. A number of published findings lead us to cautiously speculate that this system may play a role in controlling the action of other related cellular proteases. Thus, it is known that IL-1ß maturation is sometimes coupled to apoptosis 25 . Furthermore, the universal PLA2 inhibitor AACOCF3 reportedly suppresses apoptosis (26). Finally, Fas-driven apoptosis is accompanied by cleavage and inactivation of cPLA2 (27), which could tip the balance toward increased iPLA2 activity. Naturally, it cannot be excluded that other Ca2+-dependent mechanisms might also be responsible for the observed inhibitory effect of the Ca2+ ionophore on IL-1ß maturation. Future work will reveal whether subsets of cellular PLA2 are, indeed, important elements that control the functionality of other caspases.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Iwan Walev, Institute of Medical Microbiology and Hygiene, Obere Zahlbacher Strasse 67, D-55101 Mainz, Germany.
3 Abbreviations used in this paper: PLA2, phospholipase A2; iPLA2, Ca2+-independent PLA2; cPLA2, Ca2+-dependent PLA2; BEL, bromoenol lactone; GPC, glycerophosphocholine; PAP, phosphatidic acid phosphohydrolase; DAG, diacylglycerol; AA, arachidonic acid.
Received for publication May 14, 1999. Accepted for publication March 2, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and [3H]arachidonate (
) were quantified
in supernatants. Results of IL-1ß measurements are expressed in
percentage of the controls (no ionophore, no K+ depletion;
left pair of columns). Results of
[3H]arachidonate measurements are expressed as percentage
of total radioactivity in each sample (n = 3
± SD).