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and IL-1ß Occurs via a Similar Mechanism
Department of Cancer, Immunology and Infectious Diseases, Pfizer Central Research, Groton, CT 06340
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. Macrophages exposed to
ATP continuously or only for a 15-min pulse release IL-1, IL-1ß,
and lactate dehydrogenase (LDH). Proteolytic maturation of IL-1ß
exceeds that of IL-1 in both formats, but pulsed cells process the
externalized cytokines more efficiently. Ethacrynic acid and DIDS
(4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid) block ATP-induced
proteolysis of pro-IL-1ß and prevent release of pro-IL-1/ß and
LDH; they do not inhibit ATP-induced K+
(86Rb+) efflux. Ethacrynic acid inhibits
release of both forms of IL-1 with a similar concentration dependence;
within the arrested cells, procytokines accumulate in a
Triton-insoluble fraction. An IL-1ß-converting enzyme inhibitor
blocks proteolysis of IL-1ß, but it does not prevent release of
pro-IL-1, pro-IL-1ß, or LDH. These results indicate that ATP
stimulates externalization of both IL-1 and IL-1ß. The ATP-induced
cytokine release mechanism is accompanied by cell death and requires
activity of an anion transport inhibitor-sensitive component, but this
pathway operates independently of cytokine proteolytic processing. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-1 biologic activity is derived from two distinct but related gene
products, IL-1 and IL-1ß (1). The primary translation products of
the two mouse genes encode propolypeptides with apparent molecular
masses of 31 to 35 kDa (10, 11). Pro-IL-1 can bind to receptors on
target cells and elicit a biologic response (12). However, under some
circumstances, pro-IL-1 is cleaved to yield a 17-kDa mature cytokine
species that retains biologic activity; a calpain-like protease has
been implicated in this cleavage (13, 14). In contrast, pro-IL-1ß is
a weak agonist for IL-1R, and this procytokine must be proteolytically
processed to its mature 17-kDa form to demonstrate biologic activity
(15). The protease responsible for cleavage of pro-IL-1ß,
IL-1-converting enzyme
(ICE2 or caspase I), is
the founder member of a family of cysteine proteases that cleave their
substrates at an aspartic acid residue (16, 17). Activation of some
members of this protease family occurs when cells are induced to
undergo apoptosis (18, 19). Pro-IL-1 and pro-IL-1ß do not contain
signal sequences that direct them into the secretory apparatus of the
cell (20). Rather, the newly synthesized cytokine products accumulate
within the cytosol of activated monocytes/macrophages, where they
appear to coexist with inactive forms of ICE (21, 22). In vitro studies
indicate that the efficient proteolytic processing and release of
mature IL-1 require a secondary signal in addition to the primary
stimulus (e.g., LPS) that promotes transcription and translation
(23, 24, 25). The need for a second stimulus provides an additional
checkpoint by which cells may regulate biogenesis of this important
cytokine product.
Efficient IL-1ß post-translational processing has been achieved in
vitro by treating LPS-activated monocytes and macrophages with ATP (23, 26), cytolytic T cells (23, 27), bacterial toxins (28), or
potassium-selective ionophores (25, 26, 29). Likewise, peritoneal
macrophages activated with LPS in vivo require a secondary stimulus to
initiate efficient IL-1ß maturation/release, and ATP can serve in
this capacity (30). High concentrations of LPS can promote release of
IL-1ß from freshly isolated human monocytes (24), but this response
is inefficient and is lost when these cells are maintained in culture
(31). Fewer studies have examined the mechanism of IL-1 export; in
some systems, this cytokine appears to remain associated with the
plasma membrane (32). Mature forms of both IL-1 and IL-1ß are
externalized when thioglycolate-elicited/LPS-activated peritoneal
macrophages are treated with ATP or cytolytic T cells (23). Likewise,
both IL-1 and IL-1ß are released from freshly isolated human
monocytes in response to LPS activation (33). Differences in the rates
at which the two cytokine species are externalized and in the extents
to which the released cytokines are processed, however, are observed in
both the mouse and human cell systems.
In this study, we characterize ATP’s ability to initiate
post-translational processing of IL-1 and IL-1ß from murine
peritoneal macrophages in an effort to determine whether these two
cytokine species are externalized via a similar stimulus-coupled
maturation pathway. The data indicate that both cytokines are released
in response to ATP stimulation to similar extents and at comparable
rates, but that IL-1ß is processed more efficiently and more rapidly
to its mature form than is IL-1. Moreover, release of both IL-1
and IL-1ß is blocked by inhibitors of anion transport. ATP thus
constitutes an efficient stimulus for promoting externalization of both
cytokines; this stimulus-induced export pathway requires the activity
of an anion transport inhibitor-sensitive component, but functions
independently of the state of cytokine proteolytic maturation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mouse macrophages were isolated by peritoneal lavage from C3H/HeN mice (Taconic Laboratories, German Town, NY). The lavage medium consisted of RPMI 1640 containing 100 U/ml penicillin, 100 µg/ml streptomycin, 5% FBS, 2 mM glutamine, and 25 mM HEPES, pH 7.3 (culture medium). Cells from multiple animals were pooled and collected by centrifugation, washed once with culture medium, and then seeded at a density of 1 to 2 x 106 cells/well in Natrix-coated six-well plates (Collaborative Research, Bedford, MA). After 2 h at 37°C, adherent cells were washed twice with culture medium and then cultured overnight at 37°C in 2 ml of fresh culture medium in a 5% CO2 environment.
Metabolic labeling and immunoprecipitation of IL-1
Macrophages were stimulated with 1 µg/ml Escherichia
coli LPS (serotype 055:B5 obtained from Sigma Chemical Co., St.
Louis, MO) for 75 min, then rinsed once with methionine-free MEM
medium containing 100 U/ml penicillin, 100 µg/ml streptomycin, 1%
dialyzed FBS, 5 mM NaHCO3, 1 µg/ml LPS, and 20 mM HEPES,
pH 7.3 (pulse medium). One milliliter of pulse medium containing 83
µCi/ml of [35S]methionine (Amersham Corp., Chicago, IL)
was added to each well, and the macrophages were labeled at 37°C for
1 h. Labeled cells subsequently were rinsed twice with RPMI 1640
medium containing 100 U/ml penicillin, 100 µg/ml streptomycin, 1%
FBS, 2 mM glutamine, 1 µg/ml LPS, and 25 mM HEPES, pH 7.3 (chase
medium). One milliliter of chase medium was added to each well (with or
without ATP), and the cells were chased at 37°C for the indicated
times. Media then were harvested and clarified by centrifugation to
remove cells and/or cell debris. Cellular monolayers were suspended in
1 ml of a lysis buffer composed of 1% Triton X-100, 150 mM NaCl, 25 mM
HEPES, pH 7, 0.1 mM PMSF, 1 mg/ml OVA, 1 mM iodoacetic acid, 1 µg/ml
pepstatin, and 1 µg/ml leupeptin. The clarified media samples were
adjusted to the same Triton X-100 and protease inhibitor concentrations
by addition of a concentrated stock of these reagents. After 30 min on
ice, all samples were clarified by centrifugation at 45,000 rpm for 30
min in a Beckman (Palo Alto, CA) tabletop ultracentrifuge (TLA-45
rotor), and the resulting supernatants were recovered. Where indicated,
the cell-associated Triton-insoluble pellets were suspended directly in
SDS sample buffer. IL-1ß was isolated from Triton-soluble
supernatants by immunoprecipitation, as described previously (25). Goat
anti-murine IL-1 (34) was obtained from Dr. Ivan Otterness
(Pfizer Central Research, Groton, CT). This antiserum
immunoprecipitated both a 31- and 17-kDa polypeptide from macrophage
extracts and/or conditioned medium; addition of an excess of murine
mature rIL-1 prevented recovery of both species, indicating that
they are captured specifically by the Ab. The quantity of radioactivity
associated with individual IL-1 species was determined by scanning
dried gels with an Ambis Image Analysis System (San Diego,
CA).
86Rb+ efflux assay
Macrophages (5 x 105 cells/well in Natrix-coated 24-well plates) simultaneously were incubated with 1 µg/ml LPS and 3 µCi/ml [86Rb+]Cl (Amersham Corp.) for 3 h. Monolayers then were rinsed twice with chase medium to remove non-cell-associated radioactivity. Fresh medium (0.5 ml) containing, where indicated, 4,4'-diisothiocyanato stilbene-2,2'-disulfonic acid (DIDS) or ethacrynic acid was added and the cells were preincubated for 15 min at 37°C. The preincubation media were removed and replaced with fresh media containing the same effector and, where indicated, an activating stimulus. Following an incubation at 37°C, plates were placed on ice and media immediately were harvested and clarified by centrifugation. Cells (including those recovered after clarification of the media samples) were solubilized in 0.5 ml of lysis buffer. Radioactivity associated with the media and cell extracts subsequently was determined by liquid scintillation counting, and the percentage of the total (cell + medium) released extracellularly was determined. DIDS, ethacrynic acid, ATP, UTP, and benzoyl-benzoic ATP were obtained from Sigma Chemical Co. Concentrated stock solutions of the nucleotide triphosphates were adjusted to pH 7 with NaOH before their addition to cells. YVAD-CHO was obtained from Bachem Bioscience (King of Prussia, PA).
Lactate dehydrogenase (LDH) assay
Aliquots of media samples and cell extracts were assayed for LDH using pyruvate as substrate and a colorometric pyruvate detection assay (Sigma Chemical Co.); the percentage of total (sum of cell-associated and medium) LDH released extracellularly was determined.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To establish optimum conditions for IL-1ß post-translational
processing, LPS-activated, [35S]methionine-labeled
macrophages were treated with several concentrations of ATP, after
which the distribution and state of maturation of radiolabeled cytokine
were determined. In the absence of ATP, no IL-1ß was released to the
medium following 30 or 60 min of incubation, and the cell-associated
cytokine persisted as the 35-kDa procytokine species (Fig. 1
). In the presence of ATP, however,
proteolytic processing and release of IL-1ß occurred. The
cell-associated fractions contained a significant amount of 17-kDa
IL-1ß after 30 min of continuous ATP treatment, but the 35-kDa
procytokine comprised the major intracellular species (Fig. 1A). Media fractions recovered from ATP-treated
cultures, on the other hand, contained predominantly the 17-kDa IL-1ß
species and smaller quantities of the procytokine and of a 28-kDa
species (Fig. 1B); this latter polypeptide is assumed
to result from an alternate ICE cleavage event (16). Quantities of
extracellular 17-kDa IL-1ß were similar at ATP concentrations from 2
to 10 mM, but less mature cytokine was recovered at both lower (1 mM)
and higher (100 mM) concentrations of the nucleotide triphosphate (Fig. 1B). The decline in cytokine post-translational
processing observed at the highest ATP concentration is most likely the
result of an associated increase in osmolarity (ATP used as the
disodium salt); hypertonic conditions previously were shown to inhibit
the ATP response (35).
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).
Likewise, at all ATP concentrations, release of the cytoplasmic marker
LDH was increased at 60 min relative to 30 min, suggesting that the
nucleotide triphosphate promoted cytolysis (data not shown). The
absolute amount of extracellular 17-kDa IL-1ß was not increased
significantly by the longer treatment time, despite the persistence of
pro-IL-1ß intracellularly at lower (1 to 5 mM) ATP
concentrations.
Activated macrophages were treated with a single concentration of ATP
(10 mM), and the kinetics of cytokine post-translational processing
were characterized. Within 7.5 min of ATP addition, 17-kDa IL-1ß was
observed, and at this early time, the mature cytokine was recovered
exclusively intracellularly (Fig. 2). The
absolute quantity of cell-associated 17-kDa IL-1ß increased after 15
min of ATP exposure, and at this time, a small amount of mature
cytokine also was observed extracellularly. Intracellular levels of
17-kDa IL-1ß remained elevated at the 20- and 30-min harvests, after
which quantities of this species declined (Fig. 2). In contrast,
quantities of extracellular 17-kDa IL-1ß increased progressively
throughout the time course. Extracellular levels of pro-IL-1ß
remained low and constant throughout the initial 30 min of ATP
exposure, but increased sharply at the 60-min harvest (Fig. 2). The
appearance of extracellular procytokine coincided with the appearance
of extracellular LDH (Fig. 2).
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and IL-1ß
LPS-activated, [35S]methionine-labeled cells
were treated with 5 mM ATP, after which IL-1 and IL-1ß were
immunoprecipitated separately from the same cell and media extracts.
IL-1 recovered after 20, 40, and 60 min of continuous ATP treatment
migrated as the 31-kDa procytokine species; no mature IL-1 was
detected intracellularly (data not shown) or extracellularly (Fig. 3B). After the 20-min
treatment, small quantities of pro-IL-1 were recovered in the
medium, and extracellular levels of this species were increased at the
40- and 60-min harvests. In contrast, the same cell population produced
17-kDa IL-1ß, and this species was released selectively during the
initial 20 min of ATP exposure (Fig. 3A). Both mature
and precursor forms of IL-1ß, however, were recovered extracellularly
after 40 and 60 min of ATP treatment (Fig. 3A). When
radioactivity lost from IL-1ß as a result of ICE cleavage was taken
into account, a similar overall percentage of IL-1, IL-1ß, and LDH
was found to be externalized after 60 min of continuous ATP treatment
(Fig. 3C). During the initial 20 min of ATP
treatment, however, the percentage of radiolabeled IL-1ß externalized
was greater than that of IL-1 and LDH, reflecting the selective
release of the 17-kDa species.
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In the original report describing ATP-induced IL-1
post-translational processing, thioglycolate-elicited peritoneal
macrophages were exposed to ATP for 30 min and then incubated in
ATP-free medium; cytokine release into the ATP-free medium was assessed
over a subsequent 20-h period (23). To determine whether cells treated
with a pulse of ATP differed with respect to the efficiency and/or
extent of cytokine post-translational processing relative to cells
maintained continuously in the presence of the nucleotide triphosphate,
LPS-activated/[35S]methionine-labeled macrophages
were treated with 2 mM ATP for 15 min and then chased in ATP-free
medium. Processed IL-1ß was detected in the medium of ATP-pulsed
cells after only 15 min of chase (Fig. 4A). Quantities of this
17-kDa extracellular species increased with an additional 15 min of
chase, after which levels of extracellular 17-kDa IL-1ß remained
constant (Fig. 4A). Extracellular 17-kDa IL-1ß
accounted for 55% of the total IL-1ß recovered from the macrophage
cultures at this time. Remarkably, ATP-pulsed macrophages released very
little 35-kDa pro-IL-1ß (Fig. 4A).
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also was released from ATP-pulsed macrophages. Pro-IL-1 was
detected in the medium after 15 min of chase, and levels of this
species increased with an additional 15 min of post-ATP exposure (Fig. 4B). At longer chase times, several additional faster
migrating species were detected within the extracellular IL-1
immunoprecipitates; one of these possessed an apparent molecular mass
of 17 kDa and comigrated with murine mature rIL-1. This 17-kDa
species (assuming that two of six methionines were lost as a result of
cleavage (11)) represented 31% of the recovered extracellular cytokine
at the 3-h post-ATP harvest (Table I).
Quantitation of the various cytokine species indicated that similar
percentages of radiolabeled IL-1 and IL-1ß were released from the
ATP-pulsed macrophages (Table I). The overall efficiency at which the
released cytokines were processed to the 17-kDa species, however, was
much greater for IL-1ß than IL-1 (Table I). Importantly,
ATP-pulsed macrophages also released LDH in a time-dependent manner,
and the extent of LDH release correlated with the extent of IL-1
release (Table I).
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is cleaved post-release from ATP-pulsed macrophages
The above experiment indicated that 17-kDa IL-1 appeared in the
medium subsequent to the appearance of pro-IL-1. Moreover, unlike
the case with IL-1ß, no 17-kDa IL-1 was detected intracellularly
(data not shown). These observations suggested that pro-IL-1
cleavage occurred extracellularly. To test for this,
LPS-activated/[35S]methionine-labeled macrophages
were pulsed with 2 mM ATP and then chased for 30 min in ATP-deficient
medium to allow release of pro-IL-1 (see Fig. 4). At this point,
media were collected and these either were harvested immediately or
incubated for an additional 2.5 h in the absence of cells. Other
cultures were incubated for 2.5 h in the continued presence of
macrophages. IL-1 ultimately was recovered by immunoprecipitation
from all media samples and analyzed by SDS-PAGE.
Media recovered at the 30-min post-ATP harvest contained a predominance
of 35-kDa pro-IL-1 and a small amount of the 17-kDa species (Fig. 5). Levels of extracellular 17-kDa
cytokine were increased at the 3-h harvest (Fig. 5). Importantly,
30-min post-ATP harvest medium that was incubated for an additional
2.5 h in the absence of macrophages also yielded levels of 17-kDa
IL-1 greater than those recovered from the 30-min harvest (Fig. 5).
Moreover, the quantities of the 17-kDa species produced by the
cell-free incubations were comparable with those recovered from
cultures maintained in the presence of cells. Generation of the 17-kDa
IL-1 species, therefore, did not require the continued presence of
cells, and this behavior suggests that proteolytic conversion of 35-kDa
IL-1 occurred post-externalization. Inclusion of 1 mM iodoacetic
acid within the chase medium did not prevent appearance of the 17-kDa
species (Fig. 5), indicating that a cysteine protease was not
responsible for this conversion.
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and IL-1ß post-translational processing
Both anion transport and ICE inhibitors have been reported to
affect IL-1ß post-translational processing (17, 27, 36, 37, 38). To
determine whether stimulated release of IL-1 was sensitive to these
agents, ATP-pulsed macrophages were maintained in the absence or
presence of various inhibitors. In the absence of an effector,
ATP-pulsed cells released both IL-1 and IL-1ß, and extracellular
IL-1ß was processed efficiently to the 17-kDa species (Fig. 6A); IL-1 was
recovered primarily as the 35-kDa species (Fig. 6B).
Two inhibitors of anion transport, ethacrynic acid and DIDS, inhibited
post-translational processing of both IL-1 and IL-1ß. In the
presence of either of these agents, levels of extracellular IL-1ß
were reduced dramatically (Fig. 6A). Importantly, no
intracellular accumulation of 17-kDa IL-1ß species was observed in
the presence of the inhibitors (data not shown). Thus, anion transport
inhibitors prevented proteolytic conversion of pro-IL-1ß and cytokine
release. Likewise, cells treated with ethacrynic acid or DIDS released
less 35-kDa IL-1 than did their nontreated ATP-pulsed counterparts
(Fig. 6B); quantities of the 17-kDa IL-1 species
also were reduced in the presence of the anion transport inhibitors
(Fig. 6B).
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A),
and no accumulation of 17-kDa IL-1ß was observed within
YVAD-CHO-treated cells (data not shown). Macrophages treated with the
ICE inhibitor, however, continued to export pro-IL-1ß (Fig. 6A). Likewise, these cells released pro-IL-1, and
levels of the extracellular 17-kDa IL-1 species were greater
than or equal to those recovered in the absence of the ICE inhibitor
(Fig. 6B). In the absence of an effector, 56% of the culture-associated LDH activity was recovered in the medium collected at the 3-h post-ATP harvest. The anion transport inhibitors suppressed LDH release; extracellular LDH accounted for only 17 and 12% of the culture-associated LDH activity in the presence of 10 µM ethacrynic acid or 100 µM DIDS, respectively. YVAD-CHO-treated cells, however, released 68% of their LDH content.
Ethacrynic acid’s ability to inhibit externalization of IL-1 and
IL-1ß was dose dependent (Fig. 7).
Release of mature IL-1ß and pro-IL-1 displayed similar
sensitivities to ethacrynic acid, suggesting that this agent impaired a
common step in the export of both cytokines. Overall recovery of the
radiolabeled cytokines in the presence of ethacrynic acid was reduced
relative to the quantities recovered from ATP-treated cells maintained
in the drug’s absence. To account for this loss, a pulse-chase
experiment was performed. Compared with the quantities of pro-IL-1
and pro-IL-1ß recovered immediately after a 1-h pulse labeling with
[35S]methionine, total quantities of the two procytokines
recovered after 3 h of chase in the absence of ATP were reduced;
this loss is assumed to reflect normal cytokine turnover (Table II). The presence of ethacrynic acid did
not alter this basal turnover (Table II). Macrophages treated with ATP,
on the other hand, yielded quantities of the two cytokines (sum of all
intracellular and extracellular species and corrected for loss of
methionine due to proteolytic processing) comparable with those
recovered immediately after the pulse, indicating that little cytokine
turnover occurred when cytokine post-translational processing
initiated. Macrophages treated with ATP in the presence of ethacrynic
acid, on the other hand, yielded less of the radiolabeled cytokines
than non-ATP-treated controls (Table II). A similar loss was observed
in the presence of DIDS (data not shown). The reduction in
extracellular cytokine observed in the presence of anion transport
inhibitors raised the possibility that these agents did not block the
post-translational processing mechanism, but, rather, enhanced the rate
of cytokine turnover. This possibility seems unlikely, however, because
the reduction in immunoprecipitable cytokine occurred in the absence of
a change in total cell-associated radioactivity (data not shown).
Moreover, loss of immunoprecipitable cytokine was accompanied by the
appearance of two prominent radiolabeled polypeptides within the
Triton-insoluble fraction of the cells that comigrated with the
precursor forms of IL-1 and IL-1ß (Fig. 8B). The
Triton-insoluble fraction isolated from macrophages treated with
ethacrynic acid in the absence of ATP did not possess high levels of
these two polypeptides (Fig. 8A). Likewise, the
Triton-insoluble fraction isolated from cells treated with ATP did not
contain an abundance of the 31- and 35-kDa polypeptides, although
changes in the overall pattern of radiolabeled polypeptides were
observed relative to cells maintained in the absence of ATP (Fig. 8A). Loss of immunoprecipitable IL-1/ß in the
presence of a combination of ATP and ethacrynic acid, therefore,
appears to result from partitioning of the procytokines into
Triton-insoluble complexes.
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1 mM or by benzoyl benzoic ATP (Table III); this latter agent is reported to
act as a selective agonist of the P2Z receptor (40). In
contrast, ATP concentrations 
100 µM or 5 mM UTP only modestly
enhanced 86Rb+ loss (Table III). The rapid
extensive (>90%) loss of cell-associated
86Rb+, therefore, appears to proceed via the
P2Z receptor based on: 1) the requirement for ATP
concentrations in excess of 100 µM, the inactivity of UTP, and the
agonist activity of benzoyl benzoic ATP. 86Rb+
loss from macrophages maintained in the absence of ATP remained low
throughout 15 min of chase (Table IV). In
contrast, cells maintained in the presence of 2 mM ATP released >93%
of their radioactive cation content within 5 min of ATP addition (Table IV). Neither 10 µM ethacrynic acid nor 100 µM DIDS inhibited
ATP-induced loss of 86Rb+ (Table IV).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1ß from monocytes and
macrophages (23, 26). ATP’s activity as a secretion stimulus is not
shared with other nucleotide triphosphates (26) and, as shown in this
study, requires concentrations between 2 and 10 mM to achieve maximal
processing of IL-1ß. These properties indicate that ATP acts via a
P2Z type of purinoreceptor (41, 42). A
P2X7 receptor recently was cloned that possessed properties
characteristic of the P2Z receptor (43); this novel member
of the P2X superfamily, therefore, may be responsible for
initiating IL-1 post-translational processing. New findings reported in
this study indicate that ATP stimulation of LPS-activated murine
peritoneal macrophages leads to the release of similar percentages of
IL-1, IL-1ß, and LDH, and demonstrate that export of each of these
three proteins follows a similar time course. Moreover, externalization
of all three proteins is shown to be blocked by anion transport
inhibitors. In contrast, an ICE inhibitor that blocks proteolytic
processing of IL-1ß is demonstrated not to suppress release of
pro-IL-1, pro-IL-1ß, or LDH. ATP-induced cytokine externalization,
therefore, does not require proteolytic maturation of the
propolypeptides.
Macrophage IL-1ß post-translational processing was initiated within
7.5 min of nucleotide triphosphate application. Importantly, at this
time, 17-kDa IL-1ß was observed intracellularly, but not
extracellularly, indicating that proteolytic cleavage of pro-IL-1ß
occurred within the cell before externalization of the mature cytokine.
Between 15 and 30 min of ATP treatment, levels of intracellular 17-kDa
IL-1ß remained constant, while extracellular levels progressively
increased. Failure to accumulate large quantities of cell-associated
17-kDa IL-1ß suggests that the mature cytokine is externalized soon
after its formation. During this same 30-min time period, levels of
extracellular pro-IL-1ß remained low. Elevated levels of
extracellular pro-IL-1ß and LDH activity were observed after 60 min
of continuous ATP exposure; at this time, the percentages of
extracellular LDH and radiolabeled IL-1ß were comparable. This
similarity suggests that cells that initially selectively released
17-kDa IL-1ß ultimately died and released their LDH. Likewise, the
overall percentage of radiolabeled pro-IL-1 released in response to
ATP was comparable with that observed for IL-1ß and LDH. Export of
pro-IL-1, however, initially lagged behind the release of 17-kDa
IL-1ß.
Externalization of 17-kDa IL-1ß from cells activated by a 15-min
pulse of ATP occurred in parallel with the release of LDH and
pro-IL-1. Based on the rapid release of mature IL-1ß following
ATP addition noted above, it is likely that ATP-pulsed
macrophages also released 17-kDa cytokine in the absence of LDH during
the initial phase of the pulse protocol; the design of the experiment
did not allow this to be assessed. Remarkably, ATP-pulsed macrophages
did not release significant quantities of pro-IL-1ß. In contrast,
macrophages treated continuously with concentrations of ATP >2 mM
released pro-IL-1ß after prolonged (>30 min) treatment times.
Release of pro-IL-1ß from these cells may signify that continuous
exposure to high levels of the nucleotide triphosphate leads to
aberrant cytokine processing. In vivo, extracellular ATP is expected to
exist only briefly due to ubiquitous ATPases; as such, a pulse of ATP
may be more representative of a physiologically relevant stimulus. In
both the pulse and continuous formats, ATP-treated macrophages
ultimately released a similar percentage of their IL-1, IL-1ß, and
LDH contents, suggesting that ATP-induced cytokine post-translational
processing was closely associated with cell death. Previous studies
also noted that cells subjected to prolonged activation of the
P2Z receptor were destined to die (44). Cell death induced
by ATP is characterized by both apoptotic- and necrotic-type
changes (23, 45). Not all forms of cell death are sufficient to promote
proteolytic maturation of pro-IL-1ß (25); the mechanism of death
initiated by ATP, therefore, must selectively activate IL-1
post-translational processing reactions.
IL-1 released from ATP-treated thioglycolate-elicited peritoneal
macrophages is reported to be processed efficiently to the 17-kDa
species (23). In contrast, pro-IL-1 was released from resident
peritoneal macrophages in response to ATP both in vitro and in vivo
(30). Perhaps elicited and resident macrophages differ with respect to
their content of an IL-1-processing protease. Interestingly,
pro-IL-1 released to the medium as a result of the ATP pulse
subsequently was cleaved to yield a 17-kDa species. The small
quantities of this species that were produced precluded determination
of the actual cleavage site. Previous studies indicated that calpain, a
thiol protease, is responsible for proteolytic processing of
pro-IL-1 (13, 14). Extracellular processing of the procytokine
released from ATP-treated murine macrophages, however, was not blocked
by the thiol protease inhibitor iodoacetic acid nor by an ICE
inhibitor. Thus, a nonthiol protease appears to be responsible in this
system for extracellular cleavage of pro-IL-1. In contrast to
IL-1ß, processed 17-kDa IL-1 was never observed intracellularly.
At present, we do not know whether the IL-1 protease was released
from cells or was derived from serum in the medium.
In the presence of the ICE inhibitor YVAD-CHO proteolytic maturation of
IL-1ß induced by ATP was suppressed; a similar inhibition was
observed previously (38). Macrophages treated with this ICE inhibitor,
however, released quantities of pro-IL-1ß that were comparable with
the quantities of 17-kDa cytokine released in the inhibitor’s absence.
Thus, the ICE inhibitor blocked proteolytic processing, but did not
inhibit cytokine release. Likewise, quantities of IL-1 and LDH
released by ATP-treated cells were not affected by YVAD-CHO. The
inability of YVAD-CHO to prevent LDH release is consistent with
previous suggestion that ICE is not required for ATP-induced cell death
(38). Pro-IL-1ß was shown previously to be externalized by
heat-killed Staphylococcus aureus-activated human blood in
the presence of an ICE inhibitor (17). Taken together, these
observations indicate that IL-1ß release from activated monocytes
and/or macrophages is not dependent on its state of proteolytic
processing.
Two inhibitors of anion transport, on the other hand, blocked release
of IL-1, IL-1ß, and LDH. Moreover, in the presence of these
agents, no intracellular accumulation of processed IL-1ß was
observed. When LPS-activated, 86Rb+-loaded
peritoneal macrophages were treated with ATP in the absence and
presence of ethacrynic acid or DIDS, rapid and complete release of the
radioactive cation was observed. These agents, therefore, did not
prevent binding of ATP to the P2Z receptor nor subsequent
opening of the receptor-linked ion conductance (46). We demonstrated
previously that ATP initiates a volume response in human monocytes that
is sensitive to inhibitors of anion transport (35); identity of the
affected transporter remains unknown. Ethacrynic acid and DIDS,
therefore, may block ATP-induced macrophage activation by inhibiting a
change to the intracellular ionic environment that is required for
cytokine processing/release. Remarkably, within ATP-treated/ethacrynic
acid-arrested macrophages, pro-IL-1/ß redistributed to a
Triton-insoluble fraction. The significance of this localization is
unclear, but previous studies have observed associations between IL-1
and cytoskeletal (Triton-insoluble) elements (47).
The mechanism(s) used by polypeptides lacking signal sequences to gain
their release from cells remains largely unknown. In contrast to IL-1,
leaderless polypeptides, such as basic fibroblast growth factor and
galectin, are reported to be exported in the absence of cell death (48, 49), but specific components of the transport mechanisms remain to be
identified. It seems reasonable to assume that IL-1 and IL-1ß are
released by monocytes/macrophages via a common pathway, rather than
hypothesizing that each cytokine evolved a distinct and novel export
mechanism. A previous study noted, however, that IL-1 release
occurred independently of IL-1ß; LPS-activated human monocytes
externalized significant quantities of both the precursor and mature
forms of IL-1ß before releasing pro-IL-1 (33). The experimental
system used in this previous study, however, made comparison difficult.
First, human monocytes produce low levels of IL-1 relative to
IL-1ß (50); the low abundance of IL-1 hampers quantitative
comparisons. Second, LPS was used as the sole stimulus to promote
cytokine production; LPS is an efficient activator of IL-1 synthesis,
but not of IL-1 post-translational processing (51). Murine peritoneal
macrophages, on the other hand, produce comparable quantities of
radiolabeled IL-1 and IL-1ß, and when treated with ATP, they
release a high percentage of both cytokines (23); comparison under
these conditions, therefore, is less likely to be biased by differences
in cytokine quantities and/or heterogeneity of the producing cells. In
the murine peritoneal macrophage system, ATP promoted release of a
similar percentage of IL-1, IL-1ß, and LDH. This similarity is
consistent with the notion that a cell activated by ATP responds by
releasing its entire content of cytokines and LDH. Externalization
under these conditions did not require proteolytic maturation of
IL-1 or IL-1ß, although export of 17-kDa IL-1ß preceded that of
the procytokines; selective transport of mature IL-1 species has been
observed in other systems (52).
The unusual post-translational processing requirements of IL-1 offer
several potential points of therapeutic intervention aimed at limiting
cytokine activity and, in turn, suppressing inflammation. Release of
IL-1/ß from ATP-treated cells was not dependent on cytokine
proteolytic maturation. If the ATP-coupled response is representative
of a mechanism used in vivo, then agents that block proteolysis (such
as ICE inhibitors) are not expected to prevent cytokine
externalization. In view of pro-IL-1’s biologic activity (12, 53)
and the possibility that proteolytic processing of both pro-IL-1 and
pro-IL-1ß can occur extracellularly (54, 55), release of IL-1
proforms is expected to promote an inflammatory response. On the other
hand, anion transport inhibitors prevent release of both mature and
proforms of IL-1/ß; these agents may yield more profound
anti-inflammatory effects than ICE inhibitors as a result of a
larger reduction in extracellular cytokine. Therefore, therapeutic
strategies that seek to achieve an ethacrynic acid- or DIDS-like
response are expected to provide effective control of IL-1 activity
in vivo.
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Footnotes |
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2 Abbreviations used in this paper: ICE, interleukin-1ß-converting enzyme; DIDS, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid; LDH, lactate dehydrogenase; YVAD-CHO, the tetrapeptide acetyl-tyrosine-valine-alanine-aspartic acid aldehyde.
Received for publication June 4, 1997. Accepted for publication November 6, 1997.
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administration and sublethal endotoxemia in primates. Am. J. Physiol. 261:R442. cDNA. Proc. Natl. Acad. Sci. USA 85:5649.. Proc. Natl. Acad. Sci. USA 87:5548. precursor. J. Biol. Chem. 266:12162.. J. Immunol. 147:2964.[Abstract]
precursor but not the interleukin-1ß precursor. J. Biol. Chem. 262:2941.This article has been cited by other articles:
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  U. Martin, J. Scholler, J. Gurgel, B. Renshaw, J. E. Sims, and C. A. Gabel Externalization of the Leaderless Cytokine IL-1F6 Occurs in Response to Lipopolysaccharide/ATP Activation of Transduced Bone Marrow Macrophages J. Immunol., September 15, 2009; 183(6): 4021 - 4030. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
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|
|
|
|
|
|
D. Ferrari, C. Pizzirani, E. Adinolfi, R. M. Lemoli, A. Curti, M. Idzko, E. Panther, and F. Di Virgilio The P2X7 Receptor: A Key Player in IL-1 Processing and Release J. Immunol., April 1, 2006; 176(7): 3877 - 3883. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. L. Wilson, S. E. Francis, S. K. Dower, and D. C. Crossman Secretion of Intracellular IL-1 Receptor Antagonist (Type 1) Is Dependent on P2X7 Receptor Activation J. Immunol., July 15, 2004; 173(2): 1202 - 1208. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. D. Wewers IL-1{beta}: An endosomal exit PNAS, July 13, 2004; 101(28): 10241 - 10242. [Full Text] [PDF]
|
|
|
|
|
|
R. Sluyter, A. N. Shemon, and J. S. Wiley Glu496 to Ala Polymorphism in the P2X7 Receptor Impairs ATP-Induced IL-1{beta} Release from Human Monocytes J. Immunol., March 15, 2004; 172(6): 3399 - 3405. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Gudipaty, J. Munetz, P. A. Verhoef, and G. R. Dubyak Essential role for Ca2+ in regulation of IL-1{beta} secretion by P2X7 nucleotide receptor in monocytes, macrophages, and HEK-293 cells Am J Physiol Cell Physiol, August 1, 2003; 285(2): C286 - C299. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Brough, R. A. Le Feuvre, R. D. Wheeler, N. Solovyova, S. Hilfiker, N. J. Rothwell, and A. Verkhratsky Ca2+ Stores and Ca2+ Entry Differentially Contribute to the Release of IL-1{beta} and IL-1{alpha} from Murine Macrophages J. Immunol., March 15, 2003; 170(6): 3029 - 3036. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. T. Wen, C. C. Caldwell, and A. F. Knowles Poly(ADP-ribose) Polymerase Activation and Changes in Bax Protein Expression Associated with Extracellular ATP-Mediated Apoptosis in Human Embryonic Kidney 293-P2X7 Cells Mol. Pharmacol., March 1, 2003; 63(3): 706 - 713. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. A. Le Feuvre, D. Brough, Y. Iwakura, K. Takeda, and N. J. Rothwell Priming of Macrophages with Lipopolysaccharide Potentiates P2X7-mediated Cell Death via a Caspase-1-dependent Mechanism, Independently of Cytokine Production J. Biol. Chem., January 25, 2002; 277(5): 3210 - 3218. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. G. Perregaux, P. McNiff, R. Laliberte, N. Hawryluk, H. Peurano, E. Stam, J. Eggler, R. Griffiths, M. A. Dombroski, and C. A. Gabel Identification and Characterization of a Novel Class of Interleukin-1 Post-Translational Processing Inhibitors J. Pharmacol. Exp. Ther., October 1, 2001; 299(1): 187 - 197. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Di Virgilio, P. Chiozzi, D. Ferrari, S. Falzoni, J. M. Sanz, A. Morelli, M. Torboli, G. Bolognesi, and O. R. Baricordi Nucleotide receptors: an emerging family of regulatory molecules in blood cells Blood, February 1, 2001; 97(3): 587 - 600. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. FERRARI, A. LA SALA, P. CHIOZZI, A. MORELLI, S. FALZONI, G. GIROLOMONI, M. IDZKO, S. DICHMANN, J. NORGAUER, and F. DI VIRGILIO The P2 purinergic receptors of human dendritic cells: identification and coupling to cytokine release FASEB J, December 1, 2000; 14(15): 2466 - 2476. [Abstract] [Full Text]
|
|
|
|
|
|
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|
|
|
|
|
|
B. J. Gu, W. Y. Zhang, L. J. Bendall, I. P. Chessell, G. N. Buell, and J. S. Wiley Expression of P2X7 purinoceptors on human lymphocytes and monocytes: evidence for nonfunctional P2X7 receptors Am J Physiol Cell Physiol, October 1, 2000; 279(4): C1189 - C1197. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Sanz and F. D. Virgilio Kinetics and Mechanism of ATP-Dependent IL-1{beta} Release from Microglial Cells J. Immunol., May 1, 2000; 164(9): 4893 - 4898. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. E. Laliberte, J. Eggler, and C. A. Gabel ATP Treatment of Human Monocytes Promotes Caspase-1 Maturation and Externalization J. Biol. Chem., December 24, 1999; 274(52): 36944 - 36951. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. G. Perregaux and C. A. Gabel Human monocyte stimulus-coupled IL-1beta posttranslational processing: modulation via monovalent cations Am J Physiol Cell Physiol, December 1, 1998; 275(6): C1538 - C1547. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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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) and extracellular (35e; 
) proforms, and the 17-kDa
intracellular (17i; 
) and extracellular (17e; •) mature forms; all
values were normalized to total culture-associated LDH content to
correct for cell number differences. In addition, radioactivity
associated with the 17-kDa species was multiplied by a factor of 2 to
correct for loss of [35S]methionine that resulted from
proteolytic processing. Pro-IL-1ß contains eight methionines, but
only four of these are located within the mature 17-kDa
carboxyl-terminal fragment. Release of LDH into the medium (percentage
of total) also is indicated. Each data point is an average of duplicate
determinations.
