Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106
Submitted 21 February 2003 ; accepted in final form 20 March 2003
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ABSTRACT |
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interleukin-1; calcium ion; adenosine 5'-triphosphate; P2X7 receptors
Previous reports established that perturbations of monovalent cation
homeostasis, specifically the loss of intracellular K+ and gain of
Na+, within monocytes and macrophages result in the activation of
procaspase-1 and thereby accelerate the processing and release of IL-1
(2426).
However, the regulation of IL-1
processing by perturbation of monovalent
cation homeostasis is less critical in cells that maintain high levels of the
catalytically active form of caspase-1. For example, freshly isolated human
blood monocytes primed with LPS can process and secrete significant amounts of
IL-1
even in the absence of secondary stimuli that alter Na+
and K+ fluxes (34).
Nonetheless, the rate of IL-1
secretion from these cells is markedly
enhanced by P2X7 receptor activation even in the absence of
intracellular K+ loss and Na+ gain, suggesting
additional roles for this receptor in the regulation of IL-1
secretion
independent of caspase-1 activation
(26).
The extremely rapid and highly coupled reactions that characterize
P2X7 receptor-induced IL-1 processing and secretion in
monocytes and macrophages have complicated experiments aimed at dissociation
of IL-1
secretion mechanisms from the upstream pathways of caspase-1
activation and proIL-1
cleavage. Previous studies aimed at dissociating
IL-1
secretion from IL-1
processing have used a variety of
experimental systems yielding disparate results. For example, Suttles et al.
(34) reported that
Ca2+ ionophores induce mIL-1
release from
LPS-primed human monocytes but Perregaux et al.
(24) were unable to observe
similar effects of such ionophores in mouse peritoneal macrophages; this
apparent discrepancy remains unresolved. Gardella et al.
(8,
9) found that monocyte-derived
dendritic cells stimulated with T cells could package IL-1
into vesicles
resembling late, recycling endolysosomes and that this subcellular pool of
IL-1
could be secreted via a Ca2+-dependent
mechanism. Recently, MacKenzie et al.
(18) demonstrated that
2',3'-O-(4-benzoyl) benzoyl-ATP (BzATP)-induced
IL-1
release from human THP-1 monocytes occurs concurrently with
microvesiculation, a phenomenon in which the plasma membrane sheds evaginated
vesicles (
0.5-µm diameter) within seconds of P2X7 receptor
activation. Those investigators further established that HEK-293 cells
expressing P2X7 receptors also exhibited a
Ca2+-dependent microvesiculation response when
stimulated with P2X7 receptor agonists
(18).
Given these observations, we hypothesized that P2X7 receptors
induce IL-1 secretion via Ca2+-dependent
mechanisms, independent of their role in caspase-1 activation. As an
experimental model for testing this hypothesis, we used human embryonic kidney
cells (HEK-293 cells) that stably overexpress the human P2X7
receptor as a vehicle for the transient transfection of expression plasmids
encoding the mature (mIL-1
) or precursor (proIL-1
) forms of
IL-1
. These studies demonstrated that activation of P2X7
receptors results in a rapid secretion of mIL-1
by a process(es) that is
dependent on influx of extracellular Ca2+ and a
sustained rise in cytosolic Ca2+. Experiments with THP-1
human monocytes and Bac1.2F5 murine macrophages established a similar
essential role for increased cytosolic Ca2+ in the
regulation of IL-1
release by natively expressed P2X7
receptors. Finally, comparison of basal IL-1
secretion from wild-type
HEK-293 (HEK-WT) cells vs. P2X7 receptor-overexpressing HEK-293
(HEK-P2X7) cells indicated that cell surface expression of
P2X7 receptors, even in the absence of stimulation by exogenously
added ATP, results in an enhanced release of IL-1
that can be repressed
by P2X7 receptor antagonists. These results clarify an essential
role for Ca2+ influx in P2X7 receptor-induced
IL-1
secretion and suggest an additional role for P2X7
receptors as adapter proteins in the assembly or activation of the secretory
apparatus by which IL-1
is exported from cells.
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MATERIALS AND METHODS |
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Cell culture. Human embryonic kidney (HEK-293) cells were stably
transfected with the human P2X7 receptor cDNA in the pIRES vector
(HEK-P2X7). The cells were maintained in DMEM supplemented with 10%
CS, 1% PS (100 U/ml penicillin and 100 µg/ml streptomycin), and 250
µg/ml hygromycin. Control cells (HEK-WT) were stably transfected with the
empty pIRES vector (to confer hygromycin resistance) and maintained under
similar culture conditions. THP-1 human monocytic leukemia cells were cultured
in RPMI 1640 supplemented with 10% heat-inactivated CS and 2 mM glutamine.
Undifferentiated THP-1 cells were plated onto six-well
poly-L-lysine-coated culture dishes at a density of
106/ml and then cultured in the presence of human IFN-
(1,000 U/ml) for 2 days to induce monocytic differentiation
(12). The differentiated THP-1
monocytes were then primed with LPS (100 ng/ml of E. coli LPS,
K12-LCD25) for 4 h to induce expression of proIL-1
. Bac1.2F5 murine
macrophage cells were cultured as described previously
(13). For experiments, these
cells were plated onto 12-well dishes at 106/ml and stimulated with
1 µg/ml LPS (E. coli LPS, 0111:B4) for 4 h to induce proIL-1
expression.
Transfection protocols. HEK-WT or HEK-P2X7 cells were
plated onto 12-well poly-L-lysine-coated plates at 2.5 x
105 cells/well. Twenty-four hours after plating, the cell culture
medium was replaced with fresh DMEM containing 10% CS and 1% PS. The cells
were then transfected with various combinations of cDNA expression plasmids
including 0.5 µg of mIL-1, 0.5 µg of proIL-1
, or 0.1 µg
of procaspase-1 cDNA per well with standard calcium phosphate transfection
protocols. Briefly, cDNA and 125 mM CaCl2 were mixed into 0.1 ml of
(in mM) 50 Na-HEPES, 280 NaCl, 1.5 Na2HPO4, pH 7.0, and
allowed to coprecipitate for 30 min at 23°C before transfer into the 1-ml
tissue culture volume. The cells were incubated at 37°C for 18 h after
transfection before measurements of IL-1
secretion. In all experiments,
the total amount of transfected cDNA was kept constant at 0.6 µg ·
ml-1 · well-1 with pcDNA3.1
vector.
Assay for IL-1 release. At 18 h after transfection
(HEK-P2X7 or HEK-WT cells) or 4 h after LPS stimulation (THP-1
monocytes, Bac1 macrophages), the culture medium was collected and the
adherent cells were washed twice with prewarmed (37°C) phosphate-buffered
saline (PBS). The cells were then bathed in 1 ml of HEPES-buffered saline
(HBS) containing (in mM) 130 NaCl, 5 KCl, 20 HEPES, 1.0 CaCl2, 1.0
MgCl2, and 5 glucose with 0.1 mg/ml BSA, pH 7.4
(Ca2+-Mg2+-HBS) or in 1 ml of HBS
containing (in mM) 130 NaCl, 5 KCl, 20 HEPES, 2.0 MgCl2, and 5
glucose with 0.1 mg/ml BSA, pH 7.4 (Ca2+-free
Mg2+-HBS). In some experiments (see Figs.
4 and
7), the extracellular divalent
cation content was additionally varied to include 1) 0.3 mM
CaCl2 and 1.7 mM MgCl2 or 2) 0.6 mM
CaCl2 and 1.4 mM MgCl2; 130 mM NaCl was replaced with
130 mM KCl (KCl HBS) in other experiments (see
Table 1). After washing and
transfer to test saline solutions, the cells were further stimulated or not
with various agonists (3 mM ATP, 300 µM BzATP, 100 µM ADP, 100 µM
UTP, 1 µM ionomycin, or 1 µM A-23187) for up to 60 min at 37°C. At
selected times, the conditioned medium was collected and centrifuged to pellet
any detached cells; 5- to 25-µl aliquots were assayed for IL-1
by
ELISA. The cells were washed once with PBS and lysed into 0.10.2 ml of
lysis buffer (25 mM Na-HEPES, 300 mM NaCl, 1.5 mM MgCl2, 0.2 mM
EDTA, 1% Triton X-100, 2 µg/ml leupeptin, and 100 µg/ml PMSF). One- to
five-microliter aliquots of these lysates were also assayed for
IL-1
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IL-1 ELISA. Sandwich ELISA protocols were used to
assay for IL-1
in the extracellular medium samples and cell lysates.
Briefly, a 96-well plate was coated with 1 µg/ml primary anti-human or
anti-murine IL-1
by overnight incubation at 23°C and was then
blocked with 4% BSA in PBS for 1 h. Plates were washed three times with ELISA
buffer (50 mM Tris · HCl, pH 7.5, 0.2% Tween 20). Five- to
twenty-five-microliter aliquots of medium samples or cell lysates diluted to
fifty microliters with PBS were added to the blocked wells together with fifty
microliters of the second, biotinylated anti-human or anti-murine IL-1
antibody (0.2 µg/ml). Other wells were supplemented with known amounts of
human or murine IL-1
standards. The plates were incubated at 23°C
for 2 h and subsequently washed three times with the ELISA buffer. The
captured immune complexes were further incubated with streptavidin-HRP
conjugate (0.l µg/ml), washed, and colorimetrically developed with
tetramethyl benzidine (TMB) substrate for HRP, and absorbance measurements
were taken with a Molecular Devices SoftMax Pro plate reader.
Western blot analysis. Proteins were precipitated from the
conditioned medium by addition of trichloroacetic acid (TCA; 7.5% final
concentration) and cholic acid (0.1% final concentration) to each medium
sample. The precipitated proteins were washed twice with 100% acetone to
extract residual TCA and then dissolved in SDS-PAGE sample buffer. Triton
X-100 detergent-extracted cell lysates and TCA-precipitated proteins from the
extracellular medium were separated via SDS-PAGE electrophoresis with 12% gels
and then transferred to polyvinylidene difluoride (PVDF) membranes for Western
blot analysis. IL-1 was probed with the 3ZD monoclonal antibody diluted
to 5 µg/ml, caspase-1 was probed with the sc-515 antibody diluted to 1
µg/ml, and P2X7 receptor protein was probed with Alamone
anti-P2X7 antibody diluted to 2 µg/ml.
Intracellular Ca2+ concentration measurements. Intracellular Ca2+ concentration was measured with trypsinized suspensions of HEK-293 cells loaded with fura 2 and incubated in a thermostatically controlled and stirred cuvette, exactly as described previously (14). In some experiments, the nonfluorescent Ca2+ buffer BAPTA-AM (final concentration 30 µM) was added to cells for 30 min at 37°C before experimentation.
Lactate dehydrogenase assay. HEK-P2X7 cells were
stimulated with ATP or not and incubated at 37°C for up to 60 min as
described in Assay for IL-1 release. Equivalent
proportions of extracellular medium or cell lysates were assayed for lactate
dehydrogenase (LDH) activity with a Cytotoxicity Detection Kit from Boehringer
Mannheim. All values are expressed as LDH released as a percentage of the
total LDH activity measured in combined extracellular supernatants plus whole
cell lysates.
Quantitative methods. The extracellular concentration of
mIL-1 release (in pg/ml) was calculated from ELISA-based standard curves
with known quantities of human or murine recombinant IL-1
. In some
experiments, we determined the fraction of expressed mIL-1
released to
the extracellular compartment during various test stimuli. The percent release
was calculated by expressing the measured IL-1
content in the
extracellular supernatants as a percentage of the total IL-1
protein
within both the supernatant and cell lysates. As indicated in the
Fig. 1 time course, we
consistently noted a significant amount of immunoreactive IL-1
in the
extracellular medium of transfected HEK cells (corresponding to 57% of
the total cellular IL-1
content) at the nominal zero time (t)
point of test incubations, i.e., immediately after the three washes with PBS
and transfer to the test medium. This may reflect either unintended cell lysis
or mechanical activation of the secretory machinery by the fluid shear
stresses of the PBS washes and medium transfers. This IL-1
content at
t = 0 was routinely subtracted from the total IL-1
content
measured in the extracellular medium sampled at successive time points during
unstimulated or stimulated test incubations.
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Data presentation. The pooled data for
Table 1 were obtained from
n 4 experiments with each experiment performed in duplicate.
Except where noted, Figs. 1,
2,
3,
4,
5,
6,
7,
8,
9,
10 show the results from
single representative experiments. However, all experiments were performed two
to three times with similar results.
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RESULTS |
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P2X7 receptor agonists and
Ca2+ ionophores induce similar increases in
IL-1 secretion. P2X7 receptors have high
selectivity for ATP, with most other physiological nucleotides having little
or no activity. However, BzATP is a synthetic nucleotide analog that is
10-fold more potent than ATP in activating these receptors
(10,
22). To verify that the
ATP-stimulated IL-1
secretion was due to the heterologously expressed
P2X7 receptor system, HEK-P2X7 cells were also
challenged with BzATP. Figure
2A shows that 300 µM BzATP stimulated IL-1
release to the same extent (8- to 10-fold) as 3 mM ATP. In contrast, 100 µM
UTP and 100 µM ADP, which act as maximally efficacious agonists for the
P2Y2 and P2Y1 receptors that are natively expressed by
HEK-293 cells (30), induced
only twofold increases in IL-1
secretion
(Fig. 2A) during
similar 30-min test incubations. These increases in IL-1
release
triggered by the Ca2+-mobilizing P2Y receptors were
similar in magnitude to the early burst of cytokine secretion observed during
the initial 60 s of P2X7 receptor activation
(Fig. 1B). Increasing
the test concentrations of ADP or UTP to 1 mM did not induce further increases
in IL-1
secretion (data not shown). To address the role of increased
cytosolic Ca2+ in IL-1
secretion, we compared
IL-1
secretion from HEK-P2X7 cells stimulated with ATP (3
mM), ionomycin (1 µM), or A-23187 (1 µM).
Figure 2B shows that
the A-23187-stimulated release of mIL-1
(3,000 pg ·
ml-1 · 30 min-1) was
comparable in magnitude to that produced by P2X7 receptor
activation (3,200 pg · ml-1 · 30
min-1) whereas ionomycin was a less efficacious
secretagogue (2,000 pg · ml-1 · 30
min-1). Elevating the ionomycin concentration to 10
µM did not induce a further increase in IL-1
release (data not
shown).
Reduction of extracellular Ca2+ strongly
attenuates P2X7 receptor-induced IL-1
secretion. To characterize the role of Ca2+
influx in the IL-1
release response to P2X7 receptor
activation, HEK-P2X7 cells were bathed in test media containing
increasing concentrations (0, 0.3, 0.6, and 1.0 mM) of added extracellular
CaCl2. The total concentration of extracellular divalent cations
was maintained at 2 mM by replacing the CaCl2 with equimolar
MgCl2. Consistent with the function of P2X7 receptors as
ligand-gated Ca2+ channels, the magnitude of
ATP-stimulated mIL-1
secretion was progressively reduced as the
extracellular Ca2+ was decreased from the normal level
of 1 mM down to no added Ca2+ (
10 µM background;
Fig. 3A). In the
nominally Ca2+-free medium, ATP-induced IL-1
release was reduced to near-basal levels (450 pg ·
ml-1 · 30 min-1). Western
blot analysis of the same medium samples verified that this progressive
decrease in the ATP-induced release of ELISA-reactive IL-1
protein was
due to reduced extracellular levels of the 17-kDa mIL-1
(Fig. 3B). We also
noted a progressive decrease in the basal rate of IL-1
secretion from
these HEK-P2X7 cells as the extracellular
Ca2+ levels in the bathing medium were reduced. It
should be stressed that reduction or elimination of extracellular
Ca2+ does not attenuate the ability of ATP to activate
P2X7 receptors (22,
23). Indeed, we observed that
3 mM ATP triggered a similar release of intracellular K+ from
HEK-P2X7 cells incubated in Ca2+-free or
Ca2+-containing test media (data not shown). To rule out
the possibility that increased Mg2+ (2 mM
Mg2+ in the Ca2+-free HBS vs. 1 mM
Mg2+ in standard HBS) rather than decreased
Ca2+ is affecting either P2X7 receptors or
ATP-induced IL-1
release process, we performed IL-1
release
studies in Ca2+-free HBS containing the usual 1 mM
Mg2+ and observed similar inhibition of IL-1
release (data not shown). Moreover, the use of 3 mM ATP to maximally activate
P2X7 receptors minimized changes in receptor activity due to
alterations in total divalent cation concentration.
Effects of cytosolic Ca2+ buffering on the
kinetics of P2X7 receptor-induced IL-1
secretion. To further investigate the temporal relationship between
increased cytosolic Ca2+ and the stimulation of
IL-1
release during P2X7 receptor activation,
HEK-P2X7 cells transfected with mIL-1
were additionally
loaded with 30 µM BAPTA-AM to buffer rapid changes in cytosolic free
Ca2+. Figure 4,
A and B, shows that the UTP- and ADP-induced
Ca2+ transients mediated by the
Ca2+-mobilizing P2Y receptors were effectively
eliminated by BAPTA loading. BAPTA loading also significantly retarded the
initial rate at which cytosolic Ca2+ increased in
response to P2X7 receptor activation but was unable to prevent the
sustained increase in Ca2+ that accompanied stimulation
of these receptors. This indicates that the influx of extracellular
Ca2+ through the nondesensitizing P2X7
receptor channels rapidly overwhelms the buffering capacity of BAPTA.
Comparison of the kinetics of ATP-induced IL-1
release in control vs.
BAPTA-loaded cells (Fig.
4C) revealed complete inhibition of the initial,
small-magnitude burst of IL-1
release (measured at 1 min) but only
modest reduction in the extent of cytokine secretion during the second,
sustained phase of IL-1
export. A minor diminution of basal IL-1
release was also observed in the BAPTA-loaded cells compared with their
unbuffered counterparts (Fig.
4C).
P2X7 receptor activation induces
Ca2+-dependent secretion of precursor cytokine
proIL-1 from HEK-P2X7 cells.
Previous studies reported the selective release of mIL-1
but not
proIL-1
from murine P388D1 macrophages transfected with
IL-1
expression plasmids and then stimulated with
Ca2+ ionophores
(31,
32). We designed similar
experiments to test whether the Ca2+-dependent
IL-1
secretion machinery observed in our HEK-P2X7 model
system could effectively discriminate between the mature and precursor forms
of the cytokine. Western blots of cell lysates from HEK-P2X7 cells
transfected with increasing amounts of human proIL-1
or human
procaspase-1 cDNA confirmed that these cells do not natively express
proIL-1
or its processing enzyme but can rapidly accumulate large
amounts of these proteins within 18 h after transfection
(Fig. 5). We transiently
expressed proIL-1
in HEK-P2X7 cells in the absence
(Fig. 6, A and
B) or the presence
(Fig. 6, C and
D) of cotransfected caspase-1 and then measured basal vs.
ATP-stimulated IL-1
secretion in the presence of various concentrations
of extracellular Ca2+ as described above. Both ELISA
(Fig. 6A) and Western
blot (Fig. 6B)
analysis indicated that the P2X7-induced IL-1
secretion
response was not specific for mIL-1
in that proIL-1
was also
released in a Ca2+-dependent manner. It is important to
note that the antibodies used for the ELISA measurements recognize both
proIL-1
and mature IL-1
but have a significantly higher affinity
for mIL-1
. Under routine conditions, the raw ELISA data [absorbance at
450 nm (Abs450)] were calibrated relative to ELISA signals from
standard amounts of recombinant mature IL-1
. Because of the lack of
recombinant proIL-1
standards for calibration of the ELISA in terms of
proIL-1
mass, the ELISA-based secretion data in
Fig. 6, A and
C, are expressed as relative Abs450 units.
Caspase-1-mediated processing of proIL-1 to
mIL-1
is independent of P2X7
receptor-induced changes in cytosolic Ca2+. The
experiments in Fig. 6, A and
B, indicated that the
Ca2+-dependent secretion machinery triggered by
P2X7 receptor activation can readily recognize and export the
precursor form of IL-1
in the absence of competition from mIL-1
.
Previous studies showed that ectopic expression of procaspase-1 in HEK-293
cells induces autocatalytic generation of the active form of this protease,
which then facilitates processing of coexpressed proIL-1
to mIL-1
(20). Accordingly,
HEK-P2X7 cells were cotransfected with proIL-1
and
procaspase-1 and then stimulated with 3 mM ATP under the same conditions
described for the experiments in Fig. 6,
A and B. Significantly, these cells secreted
approximately equal amounts of mature IL-1
and proIL-1
when
stimulated in the standard medium containing 1 mM extracellular
Ca2+ (Fig.
6D). The similar extracellular levels of proIL-1
and mature IL-1
contrasted with the high ratio of intracellular
proIL-1
to mature IL-1
observed in the corresponding cell lysates.
This suggests that the secretory machinery can very efficiently recognize and
export mIL-1
as it is generated via the caspase-1-mediated processing of
the much more abundant proIL-1
precursor. When these proIL-1
- and
caspase-1-expressing HEK-P2X7 cells were challenged with ATP in
media containing progressively reduced levels of extracellular
Ca2+, the export of both mIL-1
and proIL-1
decreased in parallel whereas the cell lysates were characterized by a
progressive accumulation of intracellular mIL-1
(Fig. 6D). This
enhanced accumulation of intracellular mIL-1
despite the reduced export
of mIL-1
indicated that the ability of caspase-1 to convert
proIL-1
to the readily released mIL-1
was not attenuated by the
lowering of extracellular Ca2+. This suggests a unique
role for Ca2+ in the secretion of IL-1
(mature or
precursor) but not in the upstream processing of proIL-1
that is
catalyzed by active caspase-1.
Expression of P2X7 receptors enhances
IL-1 release in absence of receptor activation by exogenous
nucleotide agonists. In the course of optimizing the HEK-P2X7
cell system for investigation of P2X7 receptor-dependent IL-1
secretion, we also analyzed the secretion of transfected IL-1
from
control HEK-293 cells that lack P2X7 receptor expression.
Figure 7A shows that
these wild-type cells (HEK-WT), as well as their P2X7
receptor-expressing counterparts, accumulate similar levels of mIL-1
or
caspase-1 when identically transfected with IL-1
or caspase-1 expression
plasmids. Figure 7B
compares the basal, ATP-stimulated, and Ca2+
ionophore-stimulated IL-1
secretion from HEK-WT and HEK-P2X7
cells measured during identical 30-min incubations in standard test medium
containing 1 mM Ca2+. As expected, stimulation of the
HEK-WT cells with ATP did not increase mIL-1
export relative to the
basal secretion rate observed in unstimulated cells. However, these
comparative analyses revealed that the basal rate of mIL-1
release from
the HEK-WT cells (
120 pg · ml-1 · 30
min-1) was fourfold lower than basal secretion measured
in the HEK-P2X7 cells (450 pg · ml-1
· 30 min-1). Although stimulation of the HEK-WT
cells with ionomycin or A-23187 elicited >10-fold increases in IL-1
secretion, the absolute magnitude of the ionophore-induced effects in these
cells was 3040% lower than corresponding responses to
Ca2+ ionophores observed in the HEK-P2X7
cells. These results indicated that the Ca2+-dependent
machinery used for the secretion of mIL-1
is a general phenotypic
characteristic of HEK-293 cells regardless of the presence or absence of
P2X7 receptors. However, these experiments also suggested that the
presence of P2X7 receptors, even in the absence of deliberate
activation by exogenously added ATP, provides a stimulatory signal to the
IL-1
export machinery that mediates basal or constitutive secretion of
this cytokine. To further analyze this apparent role of P2X7
receptors in constitutive IL-1
export, we compared the absolute amounts
of mIL-1
present in the tissue culture media conditioned by either
HEK-WT cells or HEK-P2X7 cells during the 18-h incubations after
transfection with mIL-1
expression vectors.
Figure 8A shows that
HEK-P2X7 cells constitutively secreted threefold more IL-1
than HEK-WT cells during this 18-h test period. To test whether this increased
rate of constitutive IL-1
secretion might reflect a basal or
constitutive activity of the expressed P2X7 receptors, parallel
wells of the transfected cells (both HEK-WT and HEK-P2X7) were
supplemented with the covalent P2X7 antagonist oxidized ATP (oATP)
during the last 4 h of the 18-h medium-conditioning incubation
(Fig. 8A). Inclusion
of oATP in the culture medium did not affect the overall extent of mIL-1
constitutively secreted from the HEK-WT cells but produced an
40%
decrease in the amount of IL-1
present in the medium conditioned by the
HEK-P2X7 cells. To verify that the P2X7 receptors were
maximally and irreversibly inhibited by this oATP treatment protocol, wells
containing untreated or oATP-treated HEK-P2X7 cells were
supplemented with freshly added HBS test medium after removal of the
conditioned culture medium and then challenged with or without 3 mM ATP for an
additional 30 min (Fig.
8B). Although ATP stimulation produced the usual greater
than eightfold increase in IL-1
release from the untreated
HEK-P2X7 cells, the oATP-treated cells showed no increase in
IL-1
export in response to ATP. Together, the data presented in Figs.
7 and
8 suggest a role for
P2X7 receptors as enhancers of IL-1
secretion independent of
their activation by exogenously added agonist. The nature of this nominally
constitutive action of expressed P2X7 receptors remains to be
determined.
THP-1 human monocytes secrete IL-1 via
Ca2+-dependent mechanisms. We used THP-1 human
monocytic leukemia cells as a model system to characterize the role of
Ca2+ in P2X7 receptor-dependent IL-1
release from cells that natively express and secrete this cytokine. THP-1
cells were differentiated for 2 days with 1,000 U/ml IFN-
to upregulate
P2X7 receptor expression
(12) and then primed for 4 h
with 100 ng/ml LPS to induce expression of proIL-1
. Stimulation of these
primed THP-1 monocytes with ATP induced a >10-fold increase in IL-1
release when assayed in standard Ca2+-containing saline
(Fig. 9A and
Table 1). Removal of
extracellular CaCl2 from the test medium reduced this
ATP-stimulated IL-1
secretion by
85%, whereas buffering of
intracellular Ca2+ with 30 µM BAPTA-AM produced a
50% decrease in IL-1
release. When combined, removal of
extracellular Ca2+ plus BAPTA loading inhibited
ATP-induced IL-1
secretion by 90%. Western blot analysis of the
TCA-precipitated extracellular medium indicated that ATP induced release of
both proIL-1
and mIL-1
and that removal of extracellular
Ca2+ repressed the secretion of both forms of the
cytokine (Fig. 9B). In
contrast, BAPTA loading predominantly attenuated secretion of processed
mIL-1
. Western blot analysis of the cell lysates revealed an
intracellular accumulation of mature IL-1
when the THP-1 monocytes were
stimulated with ATP in Ca2+-free test saline (Fig.
9B, bottom).
Both A-23187 and ionomycin acted as IL-1
secretagogues in the LPS-primed
THP-1 monocytes, but neither ionophore was as efficacious as activated
P2X7 receptors (Table
1).
Bac1 murine macrophages secrete IL-1 via
Ca2+-dependent mechanisms. Although
Ca2+ ionophores can stimulate IL-1
secretion from
LPS-primed human monocytes
(34) or THP-1 cells
(Table 1), previous studies
indicated that such ionophores cannot potentiate IL-1
secretion from
LPS-primed mouse macrophages
(24). Thus the general role of
Ca2+ in IL-1
secretion from macrophages (mouse or
human) remains unclear. Moreover, specific roles for
Ca2+ in the ability of P2X7 receptor to
stimulate IL-1
release from macrophages have not been evaluated. We
previously (4,
13) used the Bac1.2F5 murine
macrophage line as a model system to study multiple facets of P2X7
receptor signaling in a macrophage background. When primed with 1 µg/ml LPS
for 4 h, these cells accumulated massive amounts of proIL-1
(Fig.
10B, bottom)
but secreted very minor amounts of mature IL-1
when incubated under
basal conditions (Fig. 10;
Table 1). Indeed, the basal
rate of secretion of IL-1
from these macrophages was at least 5- to
10-fold lower than that observed in THP-1 monocytes, even though both cell
types accumulated equivalent amounts of intracellular proIL-1
in
response to LPS priming. Stimulation of the Bac1 macrophages with ATP in the
standard Ca2+-containing saline elicited a >200-fold
increase in IL-1
secretion (Fig.
10A; Table
1), with proIL-1
and mIL-1
being released in
approximately equivalent amounts (Fig.
10B). Removal of extracellular CaCl2 decreased
the total ATP-stimulated IL-1
secretion by 6070%
(Fig. 10A;
Table 1), primarily because of
reduced export of mIL-1
rather than proIL-1
(Fig. 10B). Likewise,
loading the Bac1 macrophages with the BAPTA Ca2+ buffer
before ATP stimulation produced a 5055% attenuation in total IL-1
release, with the predominant effect on secretion of the processed form of the
cytokine. The combination of BAPTA loading and removal of extracellular
Ca2+ reduced P2X7 receptor-activated
IL-1
secretion by 75%. In contrast to the HEK-P2X7 cells
(Fig. 6D) and THP-1
monocytes (Fig. 9B),
inhibition of ATP-induced IL-1
secretion by removal of extracellular
Ca2+ did not lead to an enhanced intracellular
accumulation of mature IL-1
in the Bac1 macrophages
(Fig. 10B,
bottom).
Thus, as observed in the HEK-P2X7 and THP-1 cell models,
perturbation of Ca2+ homeostasis also strongly
attenuates the ability of activated P2X7 receptors to stimulate
IL-1 secretion from macrophages. However, in contrast to our findings
with HEK-P2X7 cells and THP-1 monocytes but consistent with a
previous study by Perregaux et al.
(24), stimulation of the Bac1
macrophages with either A-23187 or ionomycin produced no detectable increase
in IL-1
secretion over the very low basal rate
(Table 1). This indicates that
increased cytosolic Ca2+ per se, in the absence of the
other ionic perturbations triggered by P2X7 receptor activation, is
not a sufficient signal for activation of the IL-1
export machinery in
macrophages. Although the P2X7 receptor-induced export of mature
IL-1
secretion is similarly Ca2+ dependent in both
THP-1 monocytes and Bac1 macrophages, the other ionic signals for regulating
production of the mature cytokine clearly differ between these inflammatory
cell types. We tested this by monitoring ATP-induced IL-1
secretion from
THP-1 or Bac1 cells under conditions that eliminate the normal transmembrane
Na+ and K+ gradients (by replacing the 130 mM NaCl in
the standard test saline with equimolar KCl). In this modified saline,
activation of the nonselective P2X7 cation channels does not result
in significantly decreased intracellular K+ or increased
Na+ but does increase cytosolic Ca2+ given
the presence of normal (1 mM) extracellular CaCl2 (data not shown).
Significantly, ATP-induced secretion of IL-1
was completely inhibited
when Bac1 macrophages were incubated in this high-K+ saline but
release of the cytokine from THP-1 monocytes was only slightly attenuated
(Table 1). Consistent with the
likely role of perturbed K+/Na+ homeostasis in the
generation of mature IL-1
, rather than the export of the processed
cytokine, incubation of HEK-P2X7 cells expressing mIL-1
in
the high-K+ test saline had no inhibitory effect on P2X7
receptor-activated IL-1
secretion in that model system (data not
shown).
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DISCUSSION |
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IL-1 lacks the secretory signal sequence, and the rate of secretion
of this cytokine from LPS-primed macrophages is slow and inefficient until
activated by a secondary stimulus such as extracellular ATP. ATP targets
P2X7 receptor cation channels that mediate influx of extracellular
Na+ and Ca2+ and efflux of intracellular
K+ (21,
33). Our studies revealed that
P2X7 receptor-dependent IL-1
secretion requires
Ca2+ influx because reduction in extracellular
Ca2+ levels markedly retarded the ATP-stimulated release
of this cytokine. Biphasic secretion of IL-1
was observed such that a
minor but rapid burst of the cytokine was observed immediately after ATP
application and this was followed by a slower yet sustained rate of IL-1
release. Activation of Ca2+-mobilizing P2Y receptors
triggered IL-1
secretion that was equal in magnitude to that of the
initial burst observed with P2X7 receptor activation. Moreover, the
total amount of secreted mIL-1
was reduced in cells loaded with the
Ca2+-chelating BAPTA buffer, such that the initial burst
of IL-1
secretion was abolished although the slower and more sustained
phase was maintained under these conditions. Activation of P2X7
receptors induced rapid and sustained elevation of cytosolic
Ca2+, whereas active P2Y receptors were unable to
sustain the elevated cytosolic Ca2+ and an initial lag
was observed in the cytosolic Ca2+ increase in
BAPTA-buffered cells. Therefore, these experiments suggest that the initial
magnitude and duration of stimulus-induced increases in intracellular
Ca2+ are correlated with the rate and extent of
IL-1
export.
The results from our study reveal an essential role for a sustained
increase in cytosolic Ca2+ for the initiation of
IL-1 secretion not only in our HEK-293 model system but also in cell
types that natively express P2X7 receptors such as THP-1 monocytes
and Bac1.2F5 murine macrophages. P2X7 receptor-induced changes in
ionic homeostasis (Na+ influx, K+ efflux) additionally
modulate proIL-1
processing
(25). Efflux of intracellular
K+ promotes procaspase-1 activation, thereby providing active
caspase-1 tetramers that accelerate the rate of mIL-1
production
(25). However, LPS priming of
freshly isolated blood monocytes is sufficient to induce processing and
secretion of IL-1
, even in the absence of secondary stimuli (e.g., ATP),
primarily because LPS stimulation alone activates procaspase-1 to an
enzymatically active form. Activation of P2X7 receptors further
potentiates the rate of procaspase-1 activation and thereby IL-1
processing and release. We observed ATP-induced IL-1
secretion from
LPS-primed THP-1 monocytes regardless of whether the cells were assayed in
test medium that allows for ATP-induced K+ efflux or not
(Table 1). These data indicate
that ATP-induced K+ efflux is not an obligatory signal for
induction of IL-1
processing in THP-1 cells or blood monocytes because
LPS priming of the cells is sufficient for accumulation of active caspase-1.
However, P2X7 receptor-mediated efflux of K+ and influx
of Ca2+ accelerate an ongoing process of cytokine
production and secretion.
In contrast to blood monocytes or THP-1 cells, macrophages stimulated with
LPS alone or in conjunction with Ca2+ ionophores neither
process nor secrete IL-1 (Table
1). A potential difference may lie in the regulation of caspase-1
activity. LPS-primed macrophages require activation of procaspase-1 (by efflux
of intracellular K+; Ref.
28) In macrophages,
P2X7 receptor stimulation both activates procaspase-1 by altering
cytosolic K+ concentrations and signals to the secretory machinery
by increasing cytosolic Ca2+. Ca2+
ionophores alone are insufficient to induce IL-1
processing, because the
loss of intracellular K+ does not occur under these conditions.
Interestingly, proIL-1
was not secreted from these cells on stimulation
with the Ca2+ ionophores whereas activation of
P2X7 receptors with ATP induced release of proIL-1
and
mIL-1
(Table 1,
Fig. 10). This suggests that
macrophages contain an additional level of regulation of IL-1
secretion
that prevents release of the procytokine without procaspase-1 activation.
Consistent with this model are the previous observations by Siders et al.
(31,
32) that transfection of
precursor or mature IL-1
into P388D1 macrophages induced
selective release of mIL-1
only and not proIL-1
. In contrast, our
results in a non-macrophage cell type (HEK-P2X7 cells) demonstrate
ATP-mediated, Ca2+-dependent release of proIL-1
.
In light of these findings, we conclude that, unlike HEK cells or fresh blood
monocytes, macrophages actively inhibit proIL-1
secretion.
One potential mechanism for a macrophage-specific regulation of IL-1
release process might involve a recently identified complex of proteins termed
the "inflammasome" by Martinon et al.
(19). Among the multiple
proteins comprising the inflammasome are several that contain CARD interaction
domains including procaspase-1. It is therefore conceivable that inflammasome
formation in macrophages is the rate-limiting step for procaspase-1 activation
and mIL-1
production. In such a model, K+ efflux triggered by
activation of P2X7 receptors may favor the formation of
procaspase-1-activating inflammasome complexes.
Previous studies (5,
25,
28) of IL-1 secretion
used cells that natively expressed P2X7 receptors and IL-1
(blood monocytes and mature macrophages) or cells that transiently expressed
IL-1
but had not been characterized for P2X7 receptor
expression (P388D1 macrophages)
(31,
32). Our use of HEK-293 cells
to study secretion of IL-1
in the presence (HEK-P2X7 cells)
or absence (HEK-WT cells) of P2X7 receptors has revealed yet
another mechanism by which P2X7 receptors enhance IL-1
secretion. Comparison of basal IL-1
secretion between HEK-WT cells and
HEK-P2X7 cells indicated that HEK-P2X7 cells secreted
fourfold greater IL-1
than HEK-WT cells. This difference in the
nominally basal rate of IL-1
secretion implies that P2X7
receptors generate signals involved in IL-1
secretion even in the
absence of exogenously added ATP. Moreover, preincubation of
HEK-P2X7 cells with oATP, a P2X7 receptor antagonist,
markedly reduced this constitutive release of mIL-1
. oATP covalently
modifies the P2X7 receptor and presumably locks the conformation of
the receptor in an inactive state. Our observations suggest that the
unoccupied P2X7 receptors may have low intrinsic activity, which is
markedly enhanced by ATP binding but reversed by interactions with antagonists
such as oATP. Grahames et al.
(10) demonstrated that
LPS-primed monocytes can also secrete IL-1
in the absence of
P2X7 receptor activation and observed that these cells did not
release endogenous stores of ATP to the extracellular medium to amounts
sufficient to activate P2X7 receptors. Moreover, addition of
soluble apyrase in the extracellular medium did not reduce the total
IL-1
secreted from LPS-activated isolated blood monocytes
(10). We also observed no
effects on IL-1
secretion when soluble apyrase was added to the
extracellular media of HEK-P2X7 cells (data not shown). These data
suggest that if endogenous ATP released from HEK-P2X7 cells is
acting via autocrine mechanisms to promote P2X7 receptor
activation, the ATP must be proximal and concentrated at the cell surface
(2) such that it remains
undetectable by soluble apyrase.
One potential mechanism for an ATP-independent but P2X7
receptor-dependent regulation of IL-1 secretion might involve physical
interaction of these receptors with the secretory apparatus by which
IL-1
is released. Using HEK-293 cells expressing recombinant
P2X7 receptors, Surprenant and colleagues
(15) demonstrated that
P2X7 receptors physically associate with multiple proteins
independent of receptor activation by added ATP. These proteins include
membrane proteins, cytoskeletal and extracellular matrix proteins, as well as
some intracellular signaling proteins
(35). These interactions may
be strictly regulated in cells that natively express P2X7 receptors
(e.g., macrophages). The exact mechanisms by which the P2X7
receptors activate or enhance the secretory pathway(s) remain to be
determined. However, our observations provide insights that suggest a unique
role of this receptor in the regulation of multiple stages of the IL-1
secretory machinery.
To date, three distinct mechanisms of IL-1 secretion have been
proposed: 1) microvesiculation, 2) exocytosis of
endolysosome-like vesicles, and 3) facilitated export via an ATP
binding cassette (ABC) transporter. Microvesiculation reflects the
Ca2+-dependent shedding of vesicles from the plasma
membrane (
0.5 µm) that has been particularly well characterized in
platelet activation. Blood platelets stimulated with
Ca2+ ionophores in the presence of extracellular
Ca2+ release these microvesicles to increase the blood
procoagulant surface area (7).
Activation of P2X7 receptors was shown to induce such microvesicle
shedding via a Ca2+-dependent mechanism in THP-1
monocytes as well as in HEK-293 cells expressing recombinant P2X7
receptors (18). Moreover,
these authors detected mIL-1
in microvesicle fractions isolated from
P2X7 receptor-stimulated THP-1 monocytes. Therefore, it is possible
that the P2X7 receptor-induced sustained rise in cytosolic
Ca2+ may signal the cytoskeletal effectors that recruit,
assemble, and induce release of microvesicles and their contents. A second
mechanism for IL-1
secretion posits that IL-1
can be accumulated
into endolysosome-related vesicles that are then released by standard
exocytotic fusion with the plasma membrane. Using human monocytes, Andrei et
al. (1) observed that
proIL-1
colocalized with subcellular fractions that resemble
endolysosomal vesicles. In support of this model, they also demonstrated that
secretion of IL-1
was increased by extracellular ATP and proposed that
the regulated exocytosis of these endolysosome-related vesicles (which are
present in most cells) may be a novel secretory pathway by which leaderless
proteins such as IL-1
are efficiently exported from the cell
(1,
8). Finally, Hamon et al.
(11) reported that inhibition
of ABC transporters could attenuate ATP-dependent IL-1
secretion from
mouse peritoneal macrophages. Those authors proposed a secretory pathway that
involves facilitated efflux of IL-1
across the plasma membrane by as yet
uncharacterized ABC-type transporters. Our experiments show that, similar to
IL-1
secretion in monocytes and macrophages, activation of the
P2X7 receptors induced IL-1
export via
Ca2+-dependent mechanism(s) in cells of
non-monocytic/macrophagic origin (HEK-293 cells). This implies that the
Ca2+-dependent secretory pathway/apparatus for
IL-1
release may be ubiquitously expressed and is not unique to the
monocytes/macrophages that natively express and secrete IL-1
.
In conclusion, the proposed mechanisms of IL-1 secretion involve
either unique pathways of vesicular transport or the use of specific transport
proteins to facilitate the export of this leaderless protein. It should be
stressed that these mechanisms of transport are not mutually exclusive and
that multiple transport mechanisms may be utilized depending on cell
background and extracellular environment. For example, the initial burst of
IL-1
secretion we observed in our ATP-stimulated HEK-P2X7
cells may be mediated via prepackaged endolysosome-related vesicles that were
released on a rapid rise in cytosolic Ca2+, whereas a
more sustained rise in Ca2+ may be required for the
assembly and release of IL-1
-containing microvesicles. Regardless of the
mechanism(s) used, our studies implicate an essential role of
Ca2+ in the IL-1
export pathway that is
ubiquitously expressed.
![]() |
DISCLOSURES |
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ACKNOWLEDGMENTS |
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![]() |
FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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