Human monocyte stimulus-coupled IL-1
posttranslational
processing: modulation via monovalent cations
David G.
Perregaux and
Christopher A.
Gabel
Pfizer Central Research, Groton, Connecticut 06340
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ABSTRACT |
Lipopolysaccharide-activated human monocytes produce
prointerleukin (pro-IL)-1
but release little of this inflammatory
cytokine as the biologically active species. Efficient externalization of mature 17-kDa cytokine requires that the activated monocytes encounter a secondary stimulus such as ATP. To identify cation requirements of the ATP-induced process, lipopolysaccharide-activated monocytes were treated with ATP in media containing different Cl
salts or sucrose. Media
devoid of Na+ did not support
IL-1
processing. Titration of NaCl into choline chloride- or
sucrose-based media restored 17-kDa IL-1
production. Na+ replacement, however, was not
sufficient to support ATP-induced production of 17-kDa IL-1
in the
presence of
37 mM extracellular K+ or
Li+. Inhibition by
K+ suggests that efflux of this
cation is a necessary component of the stimulus-coupled response. The
inhibitory effect achieved by Na+
depletion is not due to inactivation of the ATP receptor and is
distinct from a caspase-1 inhibitor. Stimulus-coupled IL-1
posttranslational processing, therefore, requires extracellular Na+ for a step downstream of the
initiating stimulus but preceding caspase-1 activation.
inflammation; cytokines; P2Z
receptor
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INTRODUCTION |
INTERLEUKIN 1 (IL-1) is an important proinflammatory
mediator produced in abundance by activated monocytes and macrophages. Two distinct genes encode for IL-1 (
and
) and, despite sharing <30% sequence identity, these two cytokine products signal through shared receptors on target cells and elicit similar biologic responses (8, 43). Human IL-1
and IL-1
are produced as procytokines with
apparent molecular masses of 31 kDa (1, 23). Proteolytic activation of
pro-IL-1
occurs via caspase-1 (6, 46), the original member of a
family of cysteine proteases implicated in apoptotic processes (29,
45), to yield a 17-kDa mature biologically active product; pro-IL-1
is incompetent to bind to IL-1 receptors (25). Caspase-1 also is
required for proteolytic activation of pro-IL-18 (13). Importantly,
macrophages isolated from mice engineered to lack caspase-1 are
impaired in their production of mature IL-1
(20) and IL-18 (13).
Moreover, caspase-1-deficient mice are more resistant to
lipopolysaccharide (LPS)-induced lethality than their wild-type
counterparts (20), and inhibitors of caspase-1 limit inflammatory
cytokine production in vitro and in vivo (24, 37, 46). These
observations confirm the importance of cytokine posttranslational
processing in the generation of active inflammatory mediators. In
contrast to pro-IL-1
, pro-IL-1
is fully competent to bind to IL-1
receptors and to elicit cellular signaling pathways (25). Nonetheless,
pro-IL-1
may be proteolytically processed to a 17-kDa species, and
calpain appears to be responsible for this cleavage (5).
A remarkable feature of pro-IL-1
and pro-IL-1
is their lack of
apparent signal peptides, which are required to direct their entry into
the secretory apparatus of the cell (23). As a result, the newly
synthesized procytokines accumulate within the cytosolic compartment of
activated monocytes/macrophages (42). Previous studies indicated that
posttranslational processing of pro-IL-1
(defined as proteolytic
processing by caspase-1 and release of the mature 17-kDa species to the
medium) is an inefficient process in the absence of a secondary
stimulus (15, 16, 31, 34). Thus LPS-activated human monocytes produce
large quantities of pro-IL-1
, but the vast majority of these newly
synthesized polypeptides remains cell associated (19, 34). Release of
mature cytokine can be promoted by treating cytokine-producing cells
with high concentrations of LPS; this is an inefficient process that
appears to work best with freshly isolated monocytes (7, 18, 27). Alternatively, efficient processing can be achieved by treating LPS-activated monocytes/macrophages with extracellular ATP, cytolytic T
cells, K+-selective ionophores, or
bacterial hemolysins (3, 15, 31, 32, 35, 47). This type of
stimulus-coupled posttranslational processing is accompanied by cell
death, and the dying cells display apoptotic characteristics (14).
Although apoptosis traditionally is envisioned as a process whereby
cells are eliminated in the absence of an inflammatory response (22),
IL-1 release from dying monocytes/macrophages is expected to promote
inflammatory processes. Importantly, not all treatments that cause
monocyte/macrophage death are sufficient to promote cytokine
posttranslational processing (15, 31). The stimulus-coupled mechanism
thus is an active, rather than a passive, process.
ATP-induced IL-1
posttranslational processing is mediated via the
P2Z
(P2X7) receptor; this
designation is based on the high concentrations of ATP required to
activate the cellular response (
1 mM) and the nature of analogs that
can substitute for ATP (9, 19). Ligation of the
P2Z receptor leads to major
changes in the levels of intracellular ionic components as a result of the opening of porelike channels within the membrane (2, 4, 28, 44).
The attendant ionic changes appear to be required for efficient IL-1
posttranslational processing, and treatments that are expected to alter
ionic movements impair the cytokine response. For example, replacement
of extracellular NaCl with KCl blocks ATP-induced cytokine
posttranslational processing (32). Likewise, substitution of
extracellular Cl
with
chaotropic anions (e.g., I
or SCN
) or inclusion of
inhibitors of anion transport can block stimulus-coupled IL-1
processing (14, 19, 32, 34). Together, these observations suggest that
K+ and anion movements must occur
to facilitate the cytokine response. Consistent with this hypothesis is
the observation that hypotonic stress, a treatment known to promote
K+-Cl
efflux, activates IL-1
posttranslational processing (34, 47). In
this study we investigate the ionic requirements for stimulus-coupled processing in more detail. The results indicate that extracellular Na+ is required for efficient
ATP-induced IL-1
posttranslational processing and suggest that the
cation requirement affects a step distal to
P2Z receptor-induced pore opening.
These findings further highlight the important role of ionic changes in
monocyte/macrophage IL-1 production.
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MATERIALS AND METHODS |
Cells.
Human monocytes were isolated from heparinized blood collected from
normal volunteers. The mononuclear cell fraction prepared by
centrifugation in lymphocyte separation medium (Organon Teknika, Durham, NC) was seeded into six-well cluster dishes (1 × 107 cells/well). After 2 h of
adherence, nonattached cells were removed and discarded and the
adherent monocytes were rinsed twice with RPMI 1640 (RPMI) medium
containing 5% fetal bovine serum (FBS; maintenance medium). These
cells subsequently were cultured overnight in 2 ml of maintenance
medium at 37°C in a 5% CO2 environment.
Metabolic labeling and immunoprecipitation.
LPS (serotype 055:B5, Sigma Chemical, St. Louis, MO) was added to the
culture medium to achieve a final concentration of 10 ng/ml. Monocytes
were activated for 2 h, then the medium was removed and the cells were
rinsed once with 2 ml of methionine-free RPMI medium, 20 mM HEPES, pH
7.3, and 1% dialyzed FBS (pulse medium). One milliliter of pulse
medium containing 83 µCi/ml of
[35S]methionine
(Amersham, Arlington Heights, IL; 1,000 Ci/mmol) was added to each
well, and the monocytes were labeled for 60 min. The pulse medium
subsequently was removed, the wells were rinsed once with an
appropriate chase medium, and 1 ml of a chase medium, in the absence or
presence of ATP (2 mM) or nigericin (20 µM), was added to initiate
posttranslational processing.
After a chase period, media and cell-associated fractions were
harvested separately and IL-1
was recovered by immunoprecipitation as previously detailed (34). The resulting immunoprecipitates were
analyzed by SDS gel electrophoresis and autoradiography. The quantity
of radioactivity associated with individual forms of IL-1
was
determined by scanning dried gels with an Ambis Image Analysis System
(San Diego, CA) or with a phosphorimager (model BAS1000 MacBAS, Fuji
Medical Systems, Stamford, CT). Lactate dehydrogenase activity
associated with media samples and detergent lysates of the cell
monolayers was determined with a pyruvate detection assay (Sigma Chemical).
86Rb+
efflux assay.
Human mononuclear cells isolated as described above were seeded into
wells of 24-well cluster plates; on the basis of a cell differential, 2 × 106 monocytes were added
per well. After 2 h of adherence, nonattached cells were removed, and
the adherent monocytes were rinsed with maintenance medium and then
incubated overnight in the same medium. Monocytes were loaded with
86Rb+
by replacing the culture medium with 0.5 ml of RPMI medium containing 5% FBS, 20 mM HEPES, pH 7.3, 10 ng/ml LPS, and 6 µCi/ml
86Rb+
(NEN, Boston, MA). After a 3-h incubation with the cation, monocytes were rinsed twice with 0.5 ml of NaCl-based minimal medium containing 1% FBS before the addition of 0.5 ml of fresh minimal medium (with or
without Na+) with or without
ATP. These cultures were incubated at 37°C for up to 10 min. To
harvest the cells, plates were placed on ice and the media samples were
collected immediately. Cell monolayers were suspended in 0.5 ml of a
lysis buffer composed of 20 mM HEPES, pH 7, 150 mM NaCl, 1% Triton
X-100, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 mM iodoacetic
acid; aliquots of the media and cell-derived extracts were analyzed for
radioactivity by liquid scintillation counting. The percentage of
86Rb+
recovered extracellularly from each culture subsequently was calculated.
Reagents.
Minimal media contained 20 mM HEPES, pH 6.9, 0.9 mM
CaCl2, 0.5 mM
MgCl2, 2.7 mM KCl, 1.5 mM
KH2PO4,
5 mM glucose, 1% FBS, and 137 mM
Cl
salt (NaCl, choline
chloride, or LiCl) or 0.25 M sucrose. Media were prepared by dilution
of concentrated stock solutions of the individual components (prepared
from commercially available solid reagents with the exception of FBS);
the pH of each was adjusted to 6.9 with KOH. A microosmometer
(Precision Systems, Natick, MA) was used to measure osmolarities of
271, 297, 284, 310, 284, and 315 mosM for RPMI medium and
the 137 mM NaCl-, LiCl-, KCl-, choline chloride- and sucrose-based
minimal media, respectively. Ethacrynic acid, ATP, and nigericin were
purchased from Sigma Chemical; a 100 mM stock solution of ATP was
prepared and adjusted to pH 7 with KOH before its use. The caspase
inhibitor acetyl-tyrosine-valine-alanine-aspartic acid aldehyde
(YVAD-CHO) was obtained from Bachem Bioscience (King of Prussia, PA).
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RESULTS |
Na+ depletion
from the medium inhibits ATP-induced formation of 17-kDa
IL-1
.
LPS-activated,
[35S]methionine-labeled
human monocytes were treated with 2 mM ATP in media containing
different ionic compositions. When maintained in normal RPMI medium,
ATP-treated monocytes exported 17-kDa IL-1
(Fig.
1A,
lanes 1 and
2); a smaller amount of 31-kDa pro-IL-1
also was recovered extracellularly. Likewise, monocytes maintained in a minimal 137 mM NaCl-containing medium exported 17-kDa
IL-1
in response to ATP (Fig. 1A, lanes
3 and 4). Levels of
17-kDa IL-1
released by cells maintained in RPMI medium or the
NaCl-containing minimal medium were comparable, suggesting that special
additives to the tissue culture medium (e.g., vitamins and amino acids)
were not necessary for the cellular response. However, minimal media in
which NaCl was replaced with LiCl, choline chloride, or KCl did not
support normal IL-1
posttranslational processing; monocytes
maintained in these media released reduced quantities of 17-kDa IL-1
and enhanced levels of the 31-kDa procytokine relative to cultures
maintained in an NaCl-based medium (Fig. 1A, lanes
5-10). The magnitude of the increase in
pro-IL-1
was not consistent between different experiments; the
reason for this is unclear. Monocytes that were treated with ATP in
0.25 M sucrose also failed to release mature cytokine (Fig.
1A, lanes 11 and 12). All minimal media contained, in
addition to the 137 mM salt (or 0.25 M sucrose), 20 mM HEPES, pH 6.9, 0.9 mM CaCl2, 0.5 mM MgCl2, 2.7 mM KCl, 1.5 mM
KH2PO4,
5 mM glucose, and 1% FBS. In all cultures, IL-1
that remained cell
associated was recovered primarily as the procytokine species (data not
shown).

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Fig. 1.
Media deficient in Na+ impair
17-kDa interleukin-1 (IL-1 ) production. Lipopolysaccharide
(LPS)-activated
[35S]methionine-labeled
monocytes were incubated for 2 h with 2 mM ATP in complete RPMI 1640 (RPMI) medium or a minimal medium containing 137 mM NaCl, LiCl, choline
chloride (choline Cl), KCl, or 250 mM sucrose. After ATP treatment,
media and cells were harvested separately and IL-1 was recovered
from each by immunoprecipitation. Immunoprecipitates were analyzed by
SDS gel electrophoresis and autoradiography.
A: autoradiogram of media samples. In
a similar experiment, monocytes were treated with ATP in LiCl minimal
medium in absence ( ) or presence (+) of 5 mM
NaHCO3.
B: autoradiogram of media-associated
IL-1 immunoprecipitates. Each condition was performed in duplicate.
Arrows, migration positions of 31-kDa pro-IL-1 polypeptide, a 28-kDa
cleavage product, and 17-kDa mature IL-1 species.
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A previous study reported that IL-1 production by LPS-activated human
monocytes required extracellular
NaHCO3; in the absence of
NaHCO3, these cells failed to
alkalinize their cytoplasm in response to LPS (30). The NaCl-based
minimal medium employed above did not contain added
HCO
3, and cells maintained in this
medium yielded as much 17-kDa IL-1
in response to ATP as cells
maintained in RPMI medium (which contains 24 mM NaHCO3). Therefore, the presence
of NaHCO3 did not appear to be required for stimulus-coupled IL-1
posttranslational processing. To
ensure that the absence of HCO
3 within
the Na+-deficient media did not
account for the failure of ATP-stimulated monocytes to release 17-kDa
IL-1
, 5 mM NaHCO3 was added to
the LiCl minimal medium. This addition enhanced extracellular levels of
pro-IL-1
but it did not lead to production of the 17-kDa cytokine species (Fig. 1B). A small amount of
a 20-kDa polypeptide also was observed in the extracellular
immunoprecipitates; identity of this species is unknown.
Membrane transporters that require extracellular
Na+ to function include the
Na+/H+
antiporter (12) and the
Na+-K+-ATPase
(40). Monocytes treated with ATP in normal RPMI medium in the presence
of 5-N,N-diethylamiloride (20 µM),
an inhibitor of
Na+/H+
antiporters (39), or ouabain (1 mM), an inhibitor of the
Na+-K+-ATPase
(40), produced quantities of extracellular IL-1
comparable to those
produced by cells maintained in the absence of these inhibitors (data
not shown).
Na+
substitution can restore IL-1
posttranslational
processing.
Monocytes maintained in the 137 mM choline chloride-based minimal
medium did not release significant quantities of radiolabeled IL-1
in the absence of ATP (Fig.
2B, lanes
11 and 12). On the other hand, cells maintained in this medium in the presence of 2 mM ATP
exported pro-IL-1
and small quantities of the mature cytokine
species (Fig. 2B, lanes 5 and
6); 17-kDa IL-1
produced by these
cultures accounted for only 13% of the quantity generated by cells
maintained in an NaCl-based medium (Table
1). Cells treated with ATP in the choline
chloride-based medium released 33% of their total
[35S]methionine-labeled
IL-1
in response to ATP (Table 1, experiment A), and those maintained in NaCl- or LiCl-based media
released 70% of their overall cytokine product (Table 1). Isotonic
media containing combinations of NaCl and choline chloride restored ATP-induced 17-kDa IL-1
production (Fig. 2B, lanes
7-10). Thus, relative to cells maintained in 137 mM NaCl-based medium, monocytes treated with ATP in 45 mM NaCl-92 mM
choline chloride or 92 mM NaCl-45 mM choline chloride produced 40 and
79% as much 17-kDa IL-1
(Table 1). Moreover, the proportion of
radiolabeled cytokine externalized increased as
Na+ was titrated into the medium
(Table 1). Independent of the medium composition, IL-1
that remained
cell associated persisted as the procytokine (Fig.
2A).

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Fig. 2.
Inhibition of mature IL-1 production by monocytes maintained in
choline chloride is reversed by
Na+. LPS-stimulated,
[35S]methionine-labeled
monocytes were maintained for 2 h in minimal media containing indicated
ionic components in absence ( ) or presence (+) of 2 mM ATP.
IL-1 subsequently was recovered by immunoprecipitation from cell and
media samples, and immunoprecipitates were analyzed by SDS gel
electrophoresis. A and
B: autoradiograms of cell and media
samples, respectively. Each condition was performed in duplicate.
Arrows, migration positions of 31-, 28-, and 17-kDa species of
IL-1 .
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Monocytes maintained in the 0.25 M sucrose-based medium released
minimal quantities of IL-1
in the absence or presence of ATP (Fig.
3, lanes
1-4). As NaCl was titrated into the
sucrose-based medium, production of 17-kDa IL-1
in response to ATP
again was restored (Fig. 3, lanes
7-10). Monocytes maintained in a medium composed
of 90 mM NaCl-83 mM sucrose yielded more 17-kDa IL-1
than did cells
maintained in the 137 mM NaCl-based media (Fig. 3, Table 1,
experiment B).

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Fig. 3.
Inhibition of mature IL-1 production by monocytes maintained in 250 mM sucrose is reversed by Na+.
LPS-stimulated,
[35S]methionine-labeled
monocytes were maintained for 3 h in minimal media composed of
indicated combinations of sucrose and NaCl in absence ( ) or
presence (+) of 2 mM ATP. IL-1 subsequently was recovered by
immunoprecipitation from cell and media samples, and immunoprecipitates
were analyzed by SDS gel electrophoresis; an autoradiogram of media
samples is shown. Each condition was performed in duplicate. Arrows,
migration position of 31-, 28-, and 17-kDa species of IL-1 .
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In contrast, Na+ replacement did
not readily overcome the inhibition evoked by
K+ and
Li+. Cells maintained in the 137 mM KCl-based medium released large quantities of pro-IL-1
in
response to ATP (Fig. 4,
lanes 3 and 4), but 17-kDa mature cytokine
production was reduced >95% (Table 1, experiment
C); cytokine release in the absence of ATP was minimal in the KCl-based medium (Fig. 4, lanes
11 and 12).
Replacement of medium K+ with
concentrations of Na+ that were
sufficient to support IL-1
posttranslational processing in the
choline- or sucrose-based media yielded little improvement in
production of 17-kDa IL-1
(Fig. 4, lanes
5-10). No 17-kDa species was recovered from
monocyte cultures treated with ATP in the presence of 37 mM
Na+-100 mM
K+ or 68.5 mM
Na+-68.5 mM
K+, respectively. Moreover, when
the medium was adjusted to 100 mM
Na+-37 mM
K+, the quantity of mature
cytokine produced represented only 6% of that generated by cells
maintained in the 137 mM NaCl-based medium (Table 1). Addition of
Na+ to the medium, however, did
decrease the total amount of radiolabeled IL-1
released in response
to ATP (Fig. 4, Table 1).

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Fig. 4.
Inhibition of mature IL-1 production by monocytes maintained in KCl
is not readily reversed by Na+.
LPS-stimulated,
[35S]methionine-labeled
monocytes were incubated for 2 h in minimal media composed of indicated
concentrations of KCl or NaCl with (+) and without ( ) 2 mM ATP.
IL-1 subsequently was recovered by immunoprecipitation from extracts
of cell and media samples, and immunoprecipitates were analyzed by SDS
gel electrophoresis; an autoradiogram of media samples is shown. Each
condition was performed in duplicate. Arrows, migration positions of
31-, 28-, and 17-kDa species of IL-1 .
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Likewise, isotonic media containing combinations of LiCl and NaCl did
not support ATP-induced IL-1
posttranslational processing. Monocytes
maintained in the 137 mM LiCl-based medium released no significant
IL-1
in the absence of ATP, but addition of the nucleotide
triphosphate stimulated externalization of the procytokine species
(Table 1, experiment D). Partial
replacement of Li+ with
Na+ caused less 31-kDa pro-IL-1
to be externalized, but formation of the 17-kDa species remained
impaired at all tested
Li+-Na+
combinations (Table 1).
Recovery of total
[35S]methionine-labeled
IL-1
after the chase period was not constant between different
culture conditions within an individual experiment (Table 1). This
variance is attributed to differences in the rate of cytokine turnover
and/or recovery of immunoprecipitable cytokine from an
immunologically latent pool (34). Differences in the absolute quantity
of radiolabeled IL-1
produced between separate experiments, on the
other hand, reflect differences in cell number and/or specific
activity of [35S]methionine
employed in the labeling reactions.
Mechanism of pro-IL-1
release from ATP-treated cells
maintained in
Na+-deficient
medium is distinct from that employed to release mature
IL-1
.
Kinetics of ATP-induced IL-1
release were compared in NaCl- and
LiCl-based minimal media. In the NaCl-based medium, extracellular 17-kDa IL-1
was observed after 30 min of treatment, and the amount of this species increased eightfold by extending the treatment time to
60 min (Fig.
5A). At
this time, extracellular radiolabeled IL-1
(sum of the mature and
procytokine species and corrected for the 2-fold loss of methionines
resulting from caspase-1 cleavage) accounted for >40% of the total
(sum of intracellular and extracellular species) radiolabeled IL-1
recovered (Fig. 5B). Relative to the ATP response observed in the presence of NaCl, release of pro-IL-1
from cells maintained in the LiCl-based medium was delayed. After 15 and 30 min of ATP treatment in the LiCl-based medium, <2% of the
total radiolabeled IL-1
was externalized, and only 12% was released
after 60 min (Fig. 5B).
Extracellular levels of IL-1
recovered from the
Na+-deficient cultures after 120 min of ATP treatment in the absence of
Na+ matched those produced by a
60-min treatment in the presence of the cation (Fig.
5B).

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Fig. 5.
Comparison of kinetics of IL-1 release in absence ( ) or
presence (+) of Na+.
LPS-stimulated,
[35S]methionine-labeled
monocytes were incubated with 2 mM ATP for indicated times in 137 mM
NaCl- or 137 mM LiCl-based minimal medium. IL-1 was recovered by
immunoprecipitation from cell and media samples, and immunoprecipitates
were analyzed by SDS gel electrophoresis.
A: autoradiogram of media samples.
B: quantity of radioactivity
associated with 31-, 28-, and 17-kDa cytokine species was used to
calculate the percentage of total IL-1 (sum of cell-associated and
media species and corrected for the 2-fold loss of radioactivity due to
conversion to 17-kDa species) that was released as a function of time
and medium composition. Each bar is average of duplicate
determinations.
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Anion transport inhibitors such as ethacrynic acid previously were
shown to prevent formation and release of 17-kDa IL-1
from
ATP-treated human monocytes (19). In the NaCl-based medium, 10 µM
ethacrynic acid blocked formation of the extracellular 17-kDa species
>90% (Fig. 6,
A and
B); release of the 31-kDa species, on the other hand, was reduced only 25%. When treated with ATP in the
choline chloride-based medium, LPS-activated monocytes released small
amounts of 17-kDa IL-1
relative to cells maintained in
NaCl-based medium (Fig. 6A).
Formation of the mature cytokine species again was inhibited >90% in
the presence of ethacrynic acid, but release of pro-IL-1
into the choline chloride medium was largely unaffected (Fig.
6B). Likewise, release of 31-kDa pro-IL-1
by LiCl-maintained monocytes was reduced by only 31% in
the presence of ethacrynic acid (Fig. 6,
A and
B).

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Fig. 6.
Differential sensitivity of ATP response to ethacrynic acid in absence
and presence of extracellular Na+.
LPS-stimulated,
[35S]methionine-labeled
monocytes were incubated for 15 min in 137 mM NaCl-based minimal medium
in absence ( ) or presence (+) of 10 µM ethacrynic acid. Cells
then were incubated for an additional 3 h in NaCl, choline chloride, or
LiCl minimal media in presence of 2 mM ATP; 10 µM ethacrynic acid
remained present in cultures that had been pretreated with this agent.
Cell-associated and media fractions were harvested separately, and
IL-1 was recovered from each by immunoprecipitation; resulting
immunoprecipitates were analyzed by SDS gel electrophoresis.
A: autoradiogram of media samples.
Each condition was performed in duplicate. Arrows, migration positions
of 31-, 28-, and 17-kDa species of IL-1 .
B: quantity of radioactivity (counts,
Cts) associated with individual extracellular 31- and 17-kDa cytokine
species in A determined using a
phosphorimager and percent inhibition (%I) caused by 10 µM
ethacrynic acid. LPS-stimulated,
[35S]methionine-labeled
monocytes maintained in 137 mM NaCl-based minimal medium were
pretreated with 10 µM ethacrynic acid and/or 100 µM
acetyl-tyrosine-valine-alanine-aspartic acid aldehyde (YVAD-CHO) for 15 min, 2 mM ATP was added to each well, and cells were incubated for an
additional 3 h. Cell-associated and media samples were harvested
separately, and IL-1 was recovered from each by immunoprecipitation.
n.d., no significant radioactivity detected. C:
autoradiogram of gel containing media samples. Arrows, migration
positions of the 31-, 28-, and 17-kDa species of IL-1 .
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The modest inhibition of pro-IL-1
release observed in the presence
of ethacrynic acid contrasted sharply with this agent's effectiveness
as an inhibitor of procytokine release from monocytes maintained in the
presence of a caspase-1 inhibitor. Relative to monocytes treated with
ATP in the absence of an inhibitor, ethacrynic acid and the caspase
inhibitor YVAD-CHO (46) blocked mature cytokine formation (Fig.
6C). In contrast to ethacrynic acid-treated monocytes, however, cells treated with the caspase inhibitor externalized elevated quantities of pro-IL-1
(Fig. 6C). Monocytes incubated
simultaneously with ethacrynic acid and YVAD-CHO released quantities of
pro-IL-1
comparable to those released by the inhibitor-free cultures
and no mature cytokine (Fig. 6C).
Na+ is
required for a step distal to the P2Z
receptor.
To explore the possibility that Na+ was required for
binding of ATP to and/or operation of the
P2Z receptor, LPS-activated, [35S]methionine-labeled
monocytes were stimulated with nigericin to promote release of mature
IL-1
. This
K+/H+-exchanging
ionophore activates IL-1
posttranslational processing in the absence
of ATP (34). Monocytes treated with 20 µM nigericin in the NaCl-based
minimal medium released large quantities of radiolabeled 17-kDa IL-1
(Fig. 7, lanes
1 and 2). In
contrast, monocytes that were treated with nigericin while maintained
in the 137 mM choline chloride- or the 250 mM sucrose-based media did
not produce 17-kDa IL-1
(Fig. 7, lanes 3, 4, 7, and 8). A minimal
medium composed of 37 mM NaCl and 100 mM choline chloride allowed
17-kDa IL-1
production in response to nigericin (Fig. 7,
lanes 5 and
6), but the quantities of mature
cytokine generated were less than that produced by cells maintained in
the presence of 137 mM NaCl.

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Fig. 7.
Nigericin-induced IL-1 posttranslational processing is inhibited by
Na+ depletion. LPS-stimulated,
[35S]methionine-labeled
monocytes were treated with 20 µM nigericin for 1 h in minimal media
containing indicated components; all media in this experiment contained
5 mM KHCO3. IL-1 released into
media was recovered by immunoprecipitation and analyzed by SDS gel
electrophoresis and autoradiography; an autoradiogram is shown. Each
condition was performed in duplicate. Arrows, migration position of
31-, 28-, and 17-kDa species of IL-1 .
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Further evidence that Na+ is
required for a step downstream of the
P2Z receptor was obtained by
examining release of the K+ analog
86Rb+
from ATP-treated cells. Ligation of the
P2Z receptor is known to promote
membrane depolarization and loss of intracellular
K+ (44). Monocytes were loaded
with
86Rb+,
then its efflux was measured in the absence and presence of ATP. In the
absence of ATP, the isotope slowly dissociated from monocytes, and this
was independent of whether the cells were maintained in an NaCl- or a
choline chloride-based medium (Fig. 8).
Addition of ATP greatly accelerated the rate of
86Rb+
efflux (Fig. 8) in Na+-containing
and Na+-depleted media (Fig. 8).
The initial rate of release was augmented in the choline chloride
medium, but after 10 min of ATP exposure a similar overall percentage
of
86Rb+
was released from cultures maintained in the absence or presence of
Na+ (Fig. 8).

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Fig. 8.
Na+ depletion does not inhibit
ATP-induced
86Rb+
efflux. LPS-stimulated
86Rb+-loaded
monocytes were incubated in a 137 mM NaCl- or a 137 mM choline
chloride-based medium in absence or presence of ATP. At indicated
times, cells and media were harvested separately, and distribution of
86Rb+
was determined. Percentage of radioactive cation released into medium
is indicated as a function of time; each data point is average of
duplicate determinations.
|
|
 |
DISCUSSION |
Results of these studies indicate that extracellular monovalent cations
serve as important regulators of stimulus-coupled IL-1
posttranslational processing. Replacement of medium
Na+ with
Li+,
K+, or choline inhibited
ATP-induced formation of 17-kDa IL-1
. Likewise, when the
extracellular ionic concentration was reduced by employing a medium
containing 0.25 M sucrose, LPS-activated monocytes failed to produce
extracellular 17-kDa IL-1
in response to ATP stimulation. The
various media replacements, however, did not yield equivalent patterns
of inhibition. Monocytes maintained in a choline chloride- or
sucrose-based medium responded to ATP as evidenced by release of
pro-IL-1
, but less total IL-1
was externalized relative to cells
maintained in a minimal NaCl medium. Importantly, exchange of
Na+ for choline restored formation
and release of mature IL-1
; partial processing was observed at 45 mM
Na+, and a near-normal processing
capacity was obtained with 92 mM extracellular
Na+. Likewise, titration of
Na+ into the sucrose medium
restored processing; yield of extracellular 17-kDa IL-1
under these
conditions often exceeded that produced in the minimal NaCl-based
medium. The enhanced yield of mature cytokine likely reflects the
reduction in extracellular
Cl
; we previously observed
that cells maintained in 137 mM sodium gluconate yielded greater levels
of 17-kDa IL-1
than cells maintained in 137 mM NaCl (34). An
accentuated outward Cl
gradient may favor the ATP response. Because
Na+ concentrations required to
restore cytokine processing were similar in choline- and sucrose-based
minimal media, it is the absence of
Na+, rather than the presence of
choline or sucrose, that is considered responsible for the inhibitory effects.
Substitution of K+ for
Na+ within the medium yielded a
different outcome. Cells maintained in a 137 mM KCl-based medium
responded to ATP and released pro-IL-1
. Backexchange with
Na+ ultimately led to formation of
the 17-kDa extracellular cytokine, but return of the cytokine response
did not correspond with the aforementioned
Na+ requirements. Thus, when the
medium contained 100 mM Na+ and 37 mM K+, production of 17-kDa
IL-1
was inhibited by 90%. Because 92 mM
Na+ was sufficient to support a
complete stimulus-coupled IL-1
posttranslational processing response
in the presence of 45 mM choline, the lingering inhibition observed in
the presence of 37 mM extracellular
K+ must reflect the elevated level
of this cation. Reducing the K+
concentration to 4.2 mM (levels found in the basal NaCl medium) allowed
normal stimulus-coupled IL-1
posttranslational processing. Previously, the impaired cytokine response observed after complete replacement of Na+ with
K+ was interpreted as evidence
that high extracellular K+
prevented depletion of this cation from intracellular pools and, in
turn, blocked cytokine posttranslational processing (32, 47). The
present observation indicating that
Na+ depletion inhibits
stimulus-coupled processing complicates this interpretation. However,
because cytokine posttranslational processing remained impaired when
the medium contained elevated K+ (37 mM) and
sufficient extracellular Na+ (100 mM) to support the ATP
response, the notion that depletion of intracellular
K+ stores is necessary for
efficient cytokine posttranslational processing remains valid. The
ability of K+ ionophores and
hypotonic stress, two treatments that mobilize intracellular
K+, to serve as alternate triggers
of IL-1 posttranslational processing provides additional evidence that
K+ depletion serves as a key
element of the cytokine response pathway (32, 34, 47). It is
interesting to note that K+
depletion also has been demonstrated to be a necessary component of the
apoptotic response (17). The similar requirement for K+ efflux supports the notion that
cytokine release is associated with a form of programmed cell death
(15).
Monocytes maintained in a 137 mM LiCl-based medium also demonstrated an
aberrant ATP responsiveness. Cells maintained in this medium released
pro-IL-1
in response to ATP, and the total quantity of IL-1
externalized was comparable to that released by cells maintained in an
NaCl-based medium. Titration of
Na+ for
Li+ led to decreased extracellular
pro-IL-1
levels, but in the presence of
45 mM
Li+ no 17-kDa IL-1
was
produced. Because this inhibition by
Li+ was observed in the presence
of Na+ concentrations (92 mM) sufficient to
support normal processing, Li+ appears to be inhibitory.
How Li+ inhibits this response is unclear, but these
cations are known to affect activity of a variety of cellular
components, including protein kinases (26) and membrane transporters
(21).
How may Na+
affect stimulus-coupled IL-1
posttranslational
processing?
Since neither 5-N,N-diethylamiloride
nor ouabain inhibited ATP-induced processing, the
Na+/H+
antiporter and the
Na+-K+-ATPase
do not appear to be the targets of the
Na+ effect. ATP binding to
macrophage surface P2Z
purinoceptors leads to major changes in the intracellular ionic
environment, resulting in a complete depolarization of the membrane
potential (4, 44). Mouse thymocytes and human sperm also are reported
to depolarize when treated with millimolar concentrations of ATP (10,
36). When these latter cell types were treated with ATP in the absence of extracellular Na+, however, the
extent of depolarization was suppressed (10, 36). Influx of
extracellular Na+ through an
ATP-induced cation channel may be required to achieve complete membrane
depolarization, and/or Na+
may be required to keep the ATP-induced channel in an open
conformation. The latter possibility seems unlikely, since monocytes
treated with ATP in the absence of
Na+ released
86Rb+
to the same extent as monocytes maintained in the presence of this
cation. If it is assumed that this cation exits through the ATP-activated P2Z pore or through
a secondary transporter activated in response to the
P2Z receptor, then the absence of
Na+ did not inhibit pore
operation. Moreover, an
Na+-requiring step unrelated to
the purinoceptor is indicated by nigericin's failure to induce IL-1
posttranslational processing in
Na+-deficient media; this
ionophore is expected to initiate cytokine processing independently of
the P2Z receptor. Finally,
P2Z receptor-induced entry of
divalent cations (Ca2+ and
Ba2+) into cells is enhanced in
the absence of extracellular Na+
(36, 48), further indicating that this monovalent cation is not
required for operation of the receptor-linked ion channel.
A previous study demonstrated that 17-kDa IL-1
is released from
cells more rapidly than 31-kDa pro-IL-1
, suggesting that a
transporter may selectively export the mature cytokine species (38).
Na+ could be required for activity
of this hypothetical translocator, but the experimental observations
are not consistent with this hypothesis. In the absence of
Na+, ATP-treated cells did not
accumulate 17-kDa IL-1
intracellularly, as would be expected if
protein transport was inhibited. The suggestion has been put forward
that conversion of the procytokine species to the 17-kDa mature form is
linked to its release from the cell (41) and, as such, inhibition of a
protein translocator would not result in an intracellular accumulation
of the mature species. This possibility, however, is not consistent
with data demonstrating a transient intracellular accumulation of the
17-kDa mature cytokine species after ATP activation (31, 33).
Alternatively, caspase-1 may require
Na+ for its activity. Release of
mature IL-1
from monocytes stimulated with staphylococcal
-toxin,
however, was reported to occur equally well in the presence and absence
of extracellular Na+ (47);
Na+ thus does not appear to be
required for proteolytic maturation of IL-1
by caspase-1. Why
-toxin-induced IL-1
release should be
Na+ insensitive is unclear.
Additional evidence that the
Na+-requiring step is unrelated to
caspase-1 activation stems from the observation that monocytes released
pro-IL-1
via an ethacrynic acid-inhibitable process in the presence
of Na+ and a caspase inhibitor but
released the procytokine in an ethacrynic acid-insensitive manner in
the absence of Na+. If
Na+ depletion simply inhibited
caspase-1, then export of pro-IL-1
in the presence of YVAD-CHO would
be expected to mimic export in
Na+-deficient medium. Therefore,
Na+ appears to be required at a step in the IL-1
posttranslational pathway that precedes caspase-1 activation and
cytokine externalization. Lack of external
Na+ recently was reported to
impair release of Ca2+ from
intracellular stores in response to ATP stimulation of rat parotid
acinar cells (11). Perhaps a rise in intracellular
Ca2+ is needed for the monocyte
response. Additional studies are required to clarify the
Na+ dependence of this novel
cytokine posttranslational processing.
Despite their inability to produce mature IL-1
, monocytes treated
with ATP in the absence of extracellular
Na+ released elevated levels of
unprocessed procytokine. This ATP-induced, Na+-independent export occurred
via a mechanism distinct from that employed to release 17-kDa IL-1
in the presence of Na+ on the
basis of 1) the differential rate of
IL-1
export between the two processes and
2) the differential sensitivity of
the two processes to ethacrynic acid. We suspect in the absence of
Na+ that ATP promotes an osmotic
imbalance within monocytes that leads to swelling and lysis. Changes to
the intracellular ionic environment that occur in the absence of
Na+, however, are not sufficient
or occur in an inappropriate order such that caspase-1 fails to
activate. As a result, pro-IL-1
is not converted to the 17-kDa
species. The inability of ethacrynic acid to suppress ATP-induced
release of pro-IL-1
in the absence of
Na+ is consistent with the notion
that the nonproteolytically processed species is released via an
aberrant (lytic) mechanism that is insensitive to pharmacological intervention.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: C. A. Gabel, Dept. of Cancer,
Immunology, and Infectious Diseases, Pfizer Central Research, Groton,
CT 06340.
Received 20 April 1998; accepted in final form 25 August 1998.
 |
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