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INTRODUCTION |
Interleukin (IL)1-1 is a
multipotential inflammatory mediator produced in abundance by activated
monocytes and macrophages (1). When released from producing cells, IL-1
binds to receptors on target cells and elicits signaling cascades
leading to the up-regulation of gene products that contribute to an
inflammatory state including matrix metalloproteinases,
cyclooxygenase-2 (Cox-2), IL-6, and cellular adhesion molecules (2-5).
Two distinct gene products, IL-1
and IL-1
, contribute to IL-1
biological activity (6, 7). The amino acid sequences of IL-1
and
IL-1
are <30% identical yet these two polypeptides bind to the
same receptors on target cells (8). Human IL-1
and IL-1
both are
initially produced as 31-kDa procytokines containing amino-terminal
extensions; these extensions subsequently are removed by proteolysis.
In the case of pro-IL-1
, the propolypeptide and its 17-kDa cleavage product display equivalent signaling activity, indicating that proteolytic cleavage is not necessary to generate a receptor-competent ligand (9). In contrast, pro-IL-1
does not bind to the signaling IL-1 receptor (9), and cleavage by caspase-1 is necessary to generate
the mature 17-kDa signaling-competent form of this cytokine (10,
11).
The two forms of IL-1 share another very unusual attribute; both
pro-IL-1
and pro-IL-1
are synthesized without a signal sequence
(7), the peptide epitope required to direct nascent polypeptides to the
endoplasmic reticulum (12). As a result, newly synthesized pro-IL-1
and pro-IL-1
accumulate within the cytoplasmic compartment of
producing cells (13) rather than being sequestered to the secretory
apparatus. Caspase-1 also is produced as a cytosol-localized proenzyme;
the 45-kDa propolypeptide must be proteolytically processed to generate
the 20- and 10-kDa subunits that constitute the mature active protease
(11, 14, 15). In activated monocytes and macrophages, therefore,
pro-IL-1
and procaspase-1 co-exist within the cytoplasm. Mechanisms
that control activation of procaspase-1, and in turn cleavage of
pro-IL-1
, are not well understood. Recent studies, however, have
provided evidence that proteolytic processing of IL-1
and release of
the mature cytokine product extracellularly do not proceed
constitutively. Rather, the post-translational processing of pro-IL-1
requires that lipopolysaccharide (LPS)-activated monocytes and/or
macrophages encounter an external stimulus that promotes activation of
procaspase-1, cleavage of pro-IL-1
, and release of the 17-kDa
cytokine (16-18). Stimuli that function in vitro to promote
IL-1 post-translational processing by LPS-activated monocytes and/or
macrophages include ATP, nigericin, cytolytic T-cells, bacterial
toxins, and hypotonic stress (19-24). This requirement for a secretion
stimulus is not restricted to cells in culture; mouse peritoneal
macrophages produce pro-IL-1
in response to intraperitoneal (ip)
injection of LPS, but release little cytokine extracellularly (25).
Subsequent ip injection of ATP, however, stimulates generation of large
quantities of extracellular mature IL-1
(25).
The mechanism by which ATP activates IL-1
post-translational
processing is believed to involve the P2X7 purinergic
receptor (17, 18, 26, 27). Like other members of the P2X receptor family, the P2X7 receptor (P2X7R) is an
ATP-gated ion channel (28-30). The P2X7R, however,
demonstrates attributes that clearly distinguish it from other members
of the family. For example, the P2X7R requires levels of
ATP in excess of 1 mM to achieve activation, whereas other
P2X receptors activate at ATP concentrations of
100 µM
(31, 32); the higher concentration requirement reflects, in part, the
preference of the P2X7R for ATP4
as its
ligand and the relatively low abundance of this species in media
containing physiological concentrations of divalent cations (e.g. Ca2+ and Mg2+). An additional
unique feature of the P2X7R is found in its conductance properties. All P2X receptors demonstrate non-selective channel-like properties following ligation, but the channels formed by the P2X7R rapidly transform to pores that allow passage of
solutes as large as 900 Da (32, 33). Molecular details of this
transformation remain to be described, but domain swapping and deletion
experiments have suggested that the carboxyl-terminal domain of the
P2X7R participates in pore complex formation (28, 29); the
carboxyl-terminal domain of the P2X7R is significantly
longer than the comparable domains in the other P2X receptors (34).
Possibly as a consequence of this pore-like activity, continuous
ligation of the P2X7 receptor for times of >15 min can
lead to cell death (35-37).
In this study we employ a genetic approach to inactivate the
P2X7R. Mice lacking the P2X7R are healthy and
fertile and demonstrate no overt phenotype. However, in contrast to
their wild-type counterparts, LPS-activated peritoneal macrophages from
P2X7R
/
animals fail to generate
mature IL-1
when challenged with ATP. This defect is not because of
an inability of the macrophages to produce pro-IL-1
but rather to an
inability of the cytokine producing cells to respond to the
purinoceptor agonist. As a consequence of their inability to produce
mature IL-1
post-ATP challenge, P2X7R
/
animals generate reduced
quantities of IL-6 relative to their wild-type controls. Therefore, the
knockout animals establish that the P2X7R is a necessary
component of ATP-induced IL-1 post-translational processing, and
demonstrate that this receptor can serve as an important element of an
inflammatory cascade mechanism.
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EXPERIMENTAL PROCEDURES |
Construction of the P2X7R Targeting Vector and
Generation of the Knockout Mice--
A cDNA probe specific to the
mouse P2X7R gene was synthesized by reverse
transcription-polymerase chain reaction using primers P2X7-F1 (5'-CGGCGTGCGTTTTGACATCCT-3') and
P2X7-R2 (5'-AGGGCCCTGCGGTTCTC-3'), which were designed
based on the published rat cDNA sequence of the
P2X7R gene (28). Total RNA isolated from the
J774 A.1 mouse monocyte/macrophage cell line was used as the template
RNA. This polymerase chain reaction product was 401 base pairs long and was cloned and sequenced to verify that it corresponded to the mouse
P2X7R gene. The probe was used to screen a
129/Sv mouse genomic library and to isolate a single positive genomic
clone. Sequence analysis of BamHI subcloned fragments
confirmed that this clone corresponded to the mouse
P2X7R gene. A targeting vector was constructed
that inserted the neomycin resistance gene from the pJNS2 plasmid
directly after the Arg505 codon, deleting from
Cys506 to Pro532, which is in the
carboxyl-terminal domain of the P2X7R gene
product (38).
129/Ola-derived E14Tg2a ES cells (39) were grown, transformed, and
screened using standard methods (40). Targeted ES cells and mice
carrying the mutant allele were identified using a probe specific to a
genomic region upstream of the targeted locus. Chimeric mice derived
from targeted ES cells were mated with B6D2 (C57 BL/6 × DBA/2 F1)
or C57BL/6 mice.
Peritoneal Macrophage Isolation--
Mouse peritoneal
macrophages were harvested by injecting 5 ml of RPMI medium containing
5% FBS into each peritoneal cavity; immediately prior to injection,
the animals were euthanized. The injected medium was dispersed
throughout the peritoneal cavity, after which a hole in the skin
covering the peritoneum was introduced and the injected fluid was
recovered with the aid of a transfer pipette. Lavage fluids from
multiple animals were pooled and the cells were collected by
centrifugation (300 × g). These cell pellets were
washed twice by centrifugation in RPMI containing 5% fetal calf serum.
A cell count was performed before the final wash.
Western Analysis--
Peritoneal macrophage cell pellets were
washed once in cavitation buffer (25 mM Hepes, pH 7, 30 mM NaCl, 1 mM EDTA, 1 mM
dithiothreitol, 1 µg/ml leupeptin, and 1 µg/ml pepstatin) by
centrifugation. Cell pellets then were suspended in 2.5 ml of
cavitation buffer, and the cells were disrupted by nitrogen cavitation
(15 min on ice at 750 psi). The resulting cell lysates were adjusted to
0.1% saponin, incubated on ice for 30 min, and cell membranes
subsequently were recovered by centrifugation (50,000 rpm for 30 min at
4 °C in a Beckman Ti70 rotor). The membrane pellet was suspended in 2 ml of cavitation buffer with the aid of a glass tube-teflon pestle
homogenizer, and an aliquot of the suspension was set aside for
analysis of total protein (Pierce, Rockford, IL). The membranes again
were collected by centrifugation after which the pellets were
suspended in 100 µl of 2× Laemmli sample buffer (41).
40 µg of protein were loaded into wells of a 4-20% Tris-glycine gel
(Novex, San Diego, CA), and after separation the proteins were
transferred to nitrocellulose. These blots were blocked overnight at
4 °C in 1× Western blocking reagent (Roche Molecular Biochemicals, Indianapolis, IN) in TBS-T (10 mM Tris, pH 8, 150 mM NaCl, 0.1% Tween 20). Blots then were incubated for
2 h at room temperature in a TBS-T solution containing a 1:200
dilution of anti-P2X7R serum (Alomone, Jerusalem, Israel)
and 1× Western blocking reagent. Blots were washed in TBS-T (three
rinses, 5 min each) and then incubated for 1 h with TBS-T
containing a 1:2000 dilution of horseradish peroxidase-conjugated
anti-rabbit IgG (New England BioLabs, Beverly, MA) and 1× Western
blocking reagent. Blots were washed in TBS-T (three rinses, 5 min
each), and then developed with Super Signal (Pierce) and imaged with a
Lumi-imager (Roche Molecular Biochemicals).
For Cox-2 Western analysis, peritoneal macrophages (in RPMI, 5% FBS)
were seeded into 6-well plates (1 × 106 cells/well)
and incubated overnight at 37 °C in a 5% CO2
environment. Cells were washed twice with RPMI, 5% FBS and then 1 ml
of medium containing 100 ng/ml LPS (type 055:B5, Sigma) was added to
each well and the cells were incubated at 37 °C for 4 h. Medium
supernatants then were discarded, the adherent cells were washed twice
with PBS, and then they were solubilized by addition of 200 µl of 2× Laemmli sample buffer; the resulting samples were boiled for 3 min. 20 µl of each sample were fractionated on a 4-20% Tris-glycine gel,
after which the proteins were transferred to nitrocellulose. These
blots were processed as described above except that the primary
antibody employed was anti-prostaglandin synthase-2 (Oxford, Oxford,
MI; 1:2000 dilution) and the secondary antibody was horseradish peroxidase-conjugated rabbit anti-IgG (New England BioLabs, 1:2000 dilution).
YoPro Yellow Uptake--
Peritoneal macrophages were washed with
isotonic medium (15 mM Hepes, pH 7.2, 135 mM
NaCl, 5 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2) by centrifugation, and the resulting
cell pellet was suspended in isotonic medium to achieve a final cell
concentration of 1 × 106 cells/ml. 50 µl of this
cell suspension then was placed into wells of a Microfluor "B"
U-bottom plate (Dynatech, Chantilly, VA), and 50 µl of 2 µM YoPro Yellow (Molecular Probes, Eugene, OR) was
introduced; the fluorescent dye was dissolved in isotonic medium. Each
well then was adjusted to 5 mM ATP or 0.0075% saponin by
addition of concentrated stock solutions of these agents. Fluorescence was monitored as a function of time at 37 °C; excitation, 450 nm; emission, 530 nm.
Stimulus-induced IL-1
Post-translational Processing in
Vitro--
Macrophages from wild-type and
P2X7R
/
animals (2 × 106
cells seeded per well of 6-well cluster plates) were stimulated with 1 µg/ml LPS for 75 min and then rinsed with 2 ml of methionine-free RPMI medium containing 100 units/ml penicillin, 100 µg/ml
streptomycin, 1% dialyzed FBS, 1 µg/ml LPS, and 25 mM
Hepes, pH 7.3 (pulse medium). One ml of pulse medium containing 83 µCi/ml of [35S]methionine (Amersham Pharmacia Biotech)
then was added to each well, and the cells were labeled at 37 °C for
1 h. These labeled cells subsequently were rinsed twice with RPMI
1640 medium containing 100 units/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 ml of chase medium
containing no effector, 5 mM ATP, or 20 µM
nigericin then was added to each well, and the cells were chased at
37 °C for 30 min. Media were harvested and clarified by
centrifugation (6000 × g for 5 min) to remove cells
and/or cell debris. Cell 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 phenylmethylsulfonyl fluoride, 1 mg/ml ovalbumin, 1 mM iodoacetic acid, 1 µg/ml pepstatin, and 1 µg/ml leupeptin. Clarified medium samples
were adjusted to the same final Triton X-100 and protease inhibitor
concentrations by addition of concentrated stocks of these reagents.
After a 30-min incubation on ice, all samples were clarified by
centrifugation at 45,000 rpm for 30 min in a TLA-45 rotor (Beckman).
The resulting supernatants were recovered and IL-1
was
immunoprecipitated from these samples using a goat anti-murine IL-1
serum obtained from Dr. Ivan Otterness (Pfizer Central Reseach, Groton,
CT). Immunoprecipitates were fractionated by SDS-gel electrophoresis;
the quantity of radioactivity associated with individual IL-1
polypeptide species was determined by scanning dried gels with a phosphorimager.
ATP-induced IL-1
Post-translational Processing in
Vivo--
Groups of mice were injected ip with 1 µg of LPS. Two
hours after this LPS injection, mice were injected ip with either 0.5 ml of 30 mM ATP (adjusted to pH 7) or PBS. Mice were
euthanized 30 min or 120 min after the ATP or PBS injection, and each
peritoneal cavity was lavaged with 3 ml of media. Individual lavages
were centrifuged, supernatants were collected and tested by ELISA for the presence of IL-1
(Amersham Pharmacia Biotech) and IL-6 (Endogen, Inc. Woburn, MA). All procedures involving mice were approved by the
Institutional Animal Care and Use Committee at Pfizer Inc.
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RESULTS |
Generation and Characterization of
P2X7R
/
Mice--
Mouse ES cells in which
the P2X7R gene was disrupted by homologous
recombination were generated using the scheme shown in Fig.
1. Integration of the targeting vector
into the mouse genome by homologous recombination results in
replacement of the region of the gene encoding Cys506 to
Pro532 with the neomycin resistance gene. ES cells
containing the mutant P2X7R allele were
identified by Southern blot analysis and used to generate the
P2X7R
506-532 mouse
line. Homozygous null animals were recovered in the F2 generation with
the expected Mendelian frequencies.
P2X7R
506-532
(P2X7R
/
) animals were viable and
fertile and could not be identified among littermates by observation
alone.

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Fig. 1.
Disruption of the P2X7R gene in
ES cells and mice. A, schematic representation of the
P2X7R locus segment, the P2X7R targeting
vector, and the targeted P2X7R allele. Filled gray
boxes indicate exons and labeled boxes indicate the
PGK-TK and PGK-Neo selection cassettes. Homologous recombination of the
targeting vector with the endogenous P2X7R gene
disrupts the carboxyl-terminal coding region of the
P2X7R gene. Relevant restriction sites are
abbreviated as follows: B, BamHI; RV,
EcoRV; K, KpnI; S,
SalI. B, detection of targeted and endogenous
P2X7R alleles by Southern blot analysis of DNA
from pups derived from a heterozygous mating. DNA was digested with
EcoRV and hybridized with the probe indicated by a
dotted line in A. This probe detects a 16-kb band
from the endogenous locus and a 10-kb band from the targeted locus.
C, total RNA was prepared from cultured bone marrow-derived
mast cells from wild-type (+/+) and
P2X7R / animals and was assessed
by Northern analysis using a cDNA probe specific for the region
that is amino-terminal to the disrupted portion of the
P2X7R gene (corresponding to amino acids
Gly313-Leu492). Equal loading of the lanes and
the integrity of the mRNA obtained from the
P2X7R / cells were confirmed by
analysis of the Northern blot with an actin specific probe.
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Northern analysis of RNA isolated from cultured bone marrow mast cells
signified that the transcript for the P2X7R was present in
wild-type (+/+) but not knockout (
/
) animals (Fig. 1C).
To further demonstrate that the mutation introduced into the
P2X7R locus resulted in loss of expression of this gene,
peritoneal macrophages obtained from wild-type or
P2X7R-deficient mice were compared by Western analysis for
the presence of the receptor polypeptide. An equivalent number of
macrophages were recovered from wild-type and
P2X7R-deficient animals, suggesting that absence of the
receptor did not alter macrophage development. Membranes isolated from
wild-type peritoneal macrophages contained a 76-kDa polypeptide that
cross-reacted with the P2X7R antiserum. The size of the
murine polypeptide is comparable with that displayed by the human
P2X7R when overexpressed in HEK293 cells (Fig.
2). In contrast, macrophage membranes
prepared from cells isolated from the P2X7R-deficient
animals did not contain a similarly sized polypeptide (Fig. 2). In
addition, no smaller cross-reacting polypeptides were observed in the
Western blot of the P2X7R
/
cells, suggesting that the mutation introduced into the
P2X7R gene did not lead to expression of a
truncated version of the receptor (Fig. 2). However, because the
antibody employed was prepared against a peptide epitope that resides
at the extreme carboxyl terminus of the receptor polypeptide, we cannot
exclude the possibility that a truncated form of the receptor lacking the entire carboxyl terminus is present and not detected by the analysis.

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Fig. 2.
Peritoneal macrophages isolated from knockout
animals lack the P2X7R. Membranes isolated from
peritoneal macrophages (Macs) were fractionated by SDS-polyacrylamide
gel electrophoresis, and the polypeptides were transferred to
nitrocellulose. The blot then was probed for the presence of the
P2X7R. Control lanes contained membrane proteins isolated
from human MRC-5 fibroblasts (Fb), which do not express
P2X7R and membranes recovered from HEK293 cells transfected
with the human P2X7R.
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A hallmark of the P2X7R is its ability to facilitate
translocation of large organic molecules such as the fluorescent dye YoPro Yellow in response to ATP activation (32, 33, 42, 43). When mouse
peritoneal macrophages isolated from wild-type animals were activated
with 5 mM ATP in the presence of extracellular YoPro
Yellow, a time-dependent increase in fluorescence intensity was observed (Fig. 3). This increase in
fluorescence results from internalization of the dye molecules followed
by their binding to DNA; when bound to DNA, their fluorescence
intensity increases (43). In the absence of ATP, no significant
increase in fluorescence intensity was observed, indicating that YoPro
Yellow is impermeable to the plasma membrane in the absence of the
nucleotide triphosphate. In contrast, addition of ATP to macrophages
isolated from P2X7R
/
animals did
not result in a time-dependent increase in fluorescence intensity (Fig. 3). Macrophages isolated from the
P2X7R
/
animals demonstrated the
same low fluorescence in the absence and presence of extracellular ATP.
In the presence of saponin, a detergent that permeabilizes the plasma
membrane, YoPro Yellow accumulates to the same extent in both wild-type
and P2X7R
/
macrophages (Fig. 3).
This demonstrates that the P2X7R mediates ATP-dependent YoPro Yellow accumulation.

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Fig. 3.
Wild-type but not knockout macrophages
accumulate YoPro Yellow in response to ATP activation.
A, peritoneal macrophages were incubated with 1 µM YoPro Yellow in the presence (+ATP) or
absence ( ATP) of 5 mM ATP, and resulting
fluorescence intensity changes were recorded as a function of time at
37 °C. B, wild-type and knockout peritoneal macrophages
were incubated with 1 µM YoPro Yellow in the presence of
0.0075% saponin, and resulting fluorescence intensity changes were
recorded as a function of time.
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Stimulus-coupled IL-1
Post-translational Processing--
Peritoneal macrophages isolated from wild-type and
P2X7R
/
animals were stimulated
with LPS and labeled with [35S]methionine. These
radiolabeled cells then were chased in the absence or presence of a
secretory stimulus, after which cells and media were harvested
separately, cells were solubilized by detergent extraction, and IL-1
was recovered from the medium and cell extracts by immunoprecipitation.
Radiolabeled 35-kDa pro-IL-1
was recovered from cell extracts
derived from both wild-type and
P2X7R
/
macrophages (Fig.
4). The amount of
[35S]methionine recovered as the cell-associated 35-kDa
polypeptide (assessed by phosphorimager analysis) after the chase in
the absence of a secretory stimulus was 43,900 PSL/LDH equivalent and
44,100 PSL/LDH equivalent, respectively, from the wild-type and
knockout macrophages. This similarity suggests that the two cell-types generated comparable amounts of pro-IL-1
in response to LPS
activation. Neither the wild-type nor the
P2X7R
/
macrophages released
radiolabeled IL-1
to the medium in the absence of a secretory
stimulus (Fig. 4).

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Fig. 4.
P2X7R deficiency abolishes
ATP-induced IL-1 post-translational processing
but does not affect nigericin-induced processes. Peritoneal
macrophages were stimulated with LPS for 90 min after which the cells
were pulse-labeled with [35S]methionine for 60 min. The
radiolabeled cells then were incubated in the absence of any effector
(CON) or presence of 5 mM ATP or 20 µM nigericin (NIG) for 30 min. Cell and media
fractions were collected separately, and IL-1 was recovered from
these samples by immunoprecipitation. The immunoprecipitates were
fractionated by SDS-polyacrylamide gel electrophoresis, and
autoradiograms of the dried gels are shown for the cell-associated
(top) and media (bottom) samples.
Arrows indicate the migration positions of the 35-kDa
pro-IL-1 , 17-kDa mature IL-1 , and a 28-kDa alternate caspase-1
cleavage fragment. Each condition was performed on duplicate
cultures.
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Treatment of LPS-activated [35S]methionine-labeled
wild-type macrophages with extracellular ATP promoted formation and
release of a 17-kDa IL-1
(Fig. 4). Cytokine that remained
cell-associated was for the most part recovered as the 35-kDa
procytokine (Fig. 4). After correcting for the 2-fold loss of
radioactivity that occurs when the 35-kDa procytokine is
proteolytically processed by caspase-1 (44), the extracellular 17-kDa
polypeptide represents 64% of the total (sum of cell-associated and
medium species) radiolabeled IL-1
recovered from these cultures
(Fig. 5). In sharp contrast, LPS-activated [35S]methionine-labeled
P2X7R
/
macrophages did not
release any radiolabeled IL-1
to the medium in response to ATP (Fig.
4). Moreover, cytokine recovered from extracts of the ATP-treated
P2X7R
/
macrophages persisted as
the 35-kDa procytokine species (Fig. 4). ATP-treated wild-type but not
P2X7R-deficient macrophages demonstrated enhanced release
of LDH relative to non-ATP treated cultures (Fig. 5B).

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Fig. 5.
P2X7R deficiency selectively
affects release of mature IL-1 .
A, regions of the gel shown in Fig. 4 containing
radiolabeled IL-1 were scanned by phosphorimager analysis, and the
sum of all species (corrected for the 2-fold loss of radioactivity that
occurs when pro-IL-1 is converted to the 17-kDa species) was
determined. The % of this total that was represented by the 17-kDa
species is indicated as a function of effector treatment. Each
bar is an average of duplicate determinations. B,
the amount of LDH released into the medium (expressed as a % of the
total culture-associated activity) is indicated as a function of
effector treatment. Each bar is an average of duplicate
determinations.
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In response to the potassium ionophore nigericin, on the other hand,
both wild-type and P2X7R
/
macrophages produced and released a 17-kDa IL-1
(Fig. 4). The extracellular mature cytokine represented 66 and 74%, respectively, of
the total [35S]methionine-labeled IL-1
recovered from
the wild-type and P2X7R
/
cultures (Fig. 5A). Moreover, both macrophage populations
released comparable quantities of LDH in response to nigericin (Fig.
5B). Thus, P2X7R
/
macrophages produce pro-IL-1
in response to LPS challenge, and they
post-translationally process this procytokine in response to nigericin
stimulation. However, as a result of the absence of the
P2X7R, these cells do not produce or release mature IL-1
in response to ATP challenge.
The P2X7R recently was implicated as a component of the
pathway by which LPS induces Cox-2 expression (45). To determine whether peritoneal macrophages isolated from
P2X7R
/
animals were
impaired in their ability to generate Cox-2 following LPS activation
relative to their wild-type counterparts, cultures of the two cell
types were stimulated with LPS and Cox-2 expression was examined by
Western analysis. In the absence of LPS, neither wild-type nor
P2X7R
/
macrophages demonstrated
the presence of Cox-2 cross-reacting polypeptides (Fig.
6). However, after LPS activation, both
macrophage populations possessed a 70-kDa immunogenic polypeptide (Fig.
6); this is the expected molecular mass of murine Cox-2 (46).

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Fig. 6.
Lack of the P2X7R does not impair
Cox-2 expression in response to LPS stimulation. Peritoneal
macrophages from wild-type
(P2X7R+/+) and knockout
(P2X7R / ) animals were incubated
for 4 h in the absence and presence of LPS, after which cellular
polypeptides were separated by SDS-polyacrylamide gel electrophoresis.
The presence of Cox-2 subsequently was probed by Western analysis. Each
condition was performed on duplicate cultures. The left lane
of the blot contains standards of the indicated molecular masses.
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Characterization of In Vivo Cytokine Production
Capabilities--
Peritoneal macrophages exposed to LPS in
vivo also require a secretion stimulus to elicit efficient
externalization of mature IL-1
(25). To determine whether absence of
the P2X7R affects IL-1 release in vivo, animals
were primed with LPS, and 2 h later they received an ip injection
of PBS with or without ATP. Peritoneal lavage fluids from these animals
then were assessed for IL-1
content by ELISA. Wild-type and
heterozygous LPS-primed animals yielded no significant IL-1
in
response to PBS (Fig. 7), but abundant
quantities of IL-1
were detected following ATP challenge (Fig. 7).
In contrast, LPS-primed P2X7R
/
animals failed to generate significant levels of IL-1
in response to
ATP challenge (Fig. 7).

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Fig. 7.
P2X7R-deficient peritoneal
macrophages fail to generate IL-1 in response
to ATP stimulation in vivo. Wild-type (+/+),
heterozygous (+/ ), and homozygous
P2X7R-deficient ( / ) mice were subjected to
three different regimens: 1) untreated, 2) ip injection of LPS followed
by a second ip injection of PBS (+LPS/PBS), 3) ip injection
of LPS followed by a second injection of ATP (+LPS/+ATP).
Following these treatments, peritoneal lavage fluids were analyzed for
IL-1 content by ELISA. The amount of IL-1 recovered (ng/ml) is
indicated as a function of treatment. Each bar is the
mean ± S.D. for five mice.
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As noted earlier, IL-1 signaling often leads to the production of other
cytokines such as IL-6 (4), and cytokine networks can be initiated
in vivo in response to IL-1 generation. For example, mice
primed with an ip injection of LPS and subsequently challenged with ATP
to promote IL-1 post-translational processing generate elevated levels
of IL-6 compared with animals challenged with phosphate-buffered saline
(25). To determine whether absence of the P2X7R altered the
efficiency of IL-6 production in this type of network response,
wild-type and P2X7R
/
animals
were compared in a two-stage production assay format. Animals were
administered a priming ip injection of LPS followed 2 h later by a
PBS or ATP challenge. Peritoneal lavage fluids subsequently were
collected at 30 and 120 min, and these were assessed for cytokine
content by ELISA. Wild-type animals primed with LPS and challenged with
PBS or ATP for 30 min yielded 0.05 ng/ml and 0.4 ng/ml of IL-1
,
respectively (Table I). When the time of
the challenge reaction was extended to 2 h, IL-1
levels declined slightly to 0.033 ng/ml in the ATP-treated animals, but levels
of IL-1
within lavage fluids recovered from PBS-challenged animals
remained at the lower limit of detection (Table I). Minimal levels of
IL-1
were recovered from
P2X7R
/
animals challenged with
PBS or ATP at both the 30 and 120 min time points (Table I). Quantities
of IL-6 generated by wild-type and P2X7R-deficient animals
in response to PBS challenge were comparable at the 30 min harvest,
representing 6 and 5 ng/ml, respectively (Table I). After 120 min of
PBS challenge, IL-6 levels declined in both sets of animals to baseline
values (Table I). After 30 min of ATP challenge, both wild-type and
P2X7R
/
animals yielded 8-9
ng/ml of IL-6; these values are slightly elevated over the quantities
of IL-6 recovered from the PBS-challenged animals at 30 min (Table I).
After 120 min of ATP challenge, on the other hand, wild-type animals
yielded higher levels of IL-6 (17 ng/ml) than the
P2X7R
/
animals (6 ng/ml) (Table
I). Although levels of IL-6 generated by the ATP-challenged mutant
animals at this time were lower than those generated by their wild-type
counterparts, they were elevated above those generated in response to
PBS challenge (6 ng/ml when challenged with ATP versus 0 ng/ml when challenged with PBS; Table I). This suggests that ATP
affects IL-6 production via both a P2X7R-dependent and -independent mechanism. No
IL-1
or IL-6 could be detected in lavage fluids obtained from
animals that were primed with saline (data not shown).
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Table I
P2X7 receptor-deficient mice generate less IL-6 in response to
LPS/ATP challenge
Wild-type (WT) and P2X7R / mice were primed with an
ip injection of LPS, and 2 hr later these animals were challenged with
an additional ip injection of PBS or ATP. At 30- and 120-min
post-challenge, separate animals were sacrificed, and peritoneal
lavages were collected; cytokine content within the clarified lavage
fluids subsequently was assessed by ELISA. Each indicated value is the
mean and where indicated S.D. of six separate animals. The limit of
detection of the ELISAs, based on comparison of the assay response to
recombinant cytokine standards, was 0.05 ng/ml for the IL-1 kit and
0.05 ng/ml for the IL-6 kit.
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DISCUSSION |
Pharmacological evidence suggests that the P2X7R has a
restricted cellular distribution and is expressed primarily on cells of
hematopoietic origin including monocytes, macrophages, mast cells,
lymphocyte populations, and dendritic cells (35, 47-51). Several other
reports suggest that expression is not exclusive to this lineage as
there is evidence of P2X7R expression on sperm (52) and
some cancer cells of non-hematopoietic lineage (53). Despite extensive
studies, the physiological function of this receptor remains unclear.
It has been reported to participate in a diverse list of cellular
activities including lymphocyte proliferation (54), fertilization (52),
giant cell formation (55), cell death (36, 37), killing of invading
mycobacteria (56), and IL-1 post-translational processing (20, 22). In the absence of specific receptor agonists and antagonists, however, P2X7R participation has generally been inferred based on
ATP concentration requirements and the use of non-selective
antagonists. Following its cloning, overexpression studies established
that the P2X7R is responsible for the ability of ATP to
promote pore formation within the plasma membrane of cells expressing
this receptor (28, 29). These pores allow passage of large organic
molecules such as YoPro Yellow and ethidium bromide, a feature not
shared with other members of the P2X family of ligand-gated ion
channels. Ligation of the P2X7R also has been associated
with activation of phospholipase D and activation of some transcription
factors (57-59). To what extent these reported activities are
manifested in vivo remains to be determined. In this study
we report the generation of a mouse line that fails to express the
P2X7R polypeptide. These P2X7R-deficient mice
are healthy and fertile. The absence of striking phenotypic traits
associated with mature knockout mice suggests that the
P2X7R is dispensable for normal development. Moreover,
homozygous male animals are fertile, suggesting that absence of the
P2X7R does not compromise sperm development and function
(52). However, although their macrophages can respond to LPS and
promote pro-IL-1
and Cox-2 expression, the receptor-deficient cells
fail to generate mature IL-1
in response to subsequent ATP
challenge. As a consequence, P2X7R-deficient animals
demonstrate an attenuated response in a cytokine signaling network
in vivo.
Several previous studies demonstrated that overexpression of the
P2X7R led to the ability of ATP to promote pore formation and fluorescent dye accumulation (28, 29). Macrophages isolated from
wild-type, but not from P2X7R
/
mice, responded to extracellular ATP and accumulated YoPro Yellow. Thus, the knockout provides the corollary to the overexpression experiments; depletion of the receptor leads to an inability to form
pores that facilitate passage of YoPro yellow. These data, however, do
not prove that the P2X7R is the actual pore-forming polypeptide. BW5147 cells express the murine P2X7R, yet
these cells do not accumulate ethidium bromide in response to ATP
challenge (60). Moreover, the marine toxin maitotoxin activates
P2X7R-like pores in cells that do not express the
P2X7R (61). These observations led to the proposal that the
P2X7R itself may not constitute the pore but, rather, may
link to and/or activate the pore-forming unit (61).
One of the most intriguing activities attributed to the
P2X7R is its ability to induce post-translational
processing of pro-IL-1 (18, 20, 22). Data previously have been
presented establishing that efficient release of mature IL-1
from
LPS-activated monocytes and macrophages requires the cytokine-producing
cells to encounter a secretion stimulus. Agents or treatments that have
been shown to function in this capacity in vitro include
ATP, nigericin, hypotonic stress, cytolytic T-cells, and bacterial
toxins (19-24). All of these effectors appear to initiate IL-1
post-translational processing reactions by inducing changes to the
intracellular ionic environment. For example, treatment of
LPS-activated monocytes with ATP or nigericin promotes loss of
intracellular K+ (22). When this loss is prevented by
exposing the cells to the secretion stimuli in the presence of elevated
extracellular K+ concentrations, IL-1
post-translational
processing is completely inhibited (18, 22, 62). Therefore, depletion
of intracellular K+ appears to be a necessary element of
the stimulus-induced process. Likewise, inhibitors of anion transport,
such as tenidap, ethacrynic acid, and glyburide, disrupt ATP-induced
IL-1
post-translational processing suggesting that anion movements
are a necessary feature of the cellular process (17, 26, 63).
Peritoneal macrophages isolated from the
P2X7R
/
mice responded to LPS
activation and produced quantities of pro-IL-1
and Cox-2 comparable
with those produced by wild-type macrophages. Although we cannot rule
out the possibility of the involvement of a compensatory mechanism,
these data suggest that the P2X7R is not required for induction of LPS-inducible gene products. In contrast to our findings, a previous study concluded that ATP released from macrophages as a
result of LPS activation served as an autocrine-type stimulus to
promote expression of Cox-2 via ligation of the P2X7R (45). Conclusions reached in this previous study, however, were based on the
use of non-selective inhibitors of the P2X7R, and these compounds may elicit their effects by disrupting other cellular processes. Although expression of pro-IL-1
was normal in macrophages that lacked the P2X7R, post-translational processing of the
procytokine in response to ATP challenge was totally ablated.
Therefore, the P2X7R is a necessary element of the
post-translational processing mechanism. Interestingly, LPS-activated
P2X7R-deficient macrophages continued to produce mature
IL-1
in response to nigericin challenge. Nigericin, therefore, must
promote cytokine post-translational processing independently of the
P2X7R. The mechanisms by which nigericin and ATP initiate
pro-IL-1
post-translational processing appear to share common
elements. For example, both mechanisms require extracellular
Na+ (62), both are inhibited by high extracellular
K+ (22), and both result in similar morphological
transformations (22). Perhaps, these two diverse stimuli link to a
common pore-forming subunit to initiate the cytokine production pathway.
IL-1 is known to elicit complex cytokine signaling networks when
administered to animals, and injection of the recombinant cytokine
locally within animal joints can lead to an inflammatory response and
attendant structural changes that mimic the pathophysiological process
taking place in joints of patients suffering from rheumatoid arthritis
(Ref. 64). Many gene products are known to be up-regulated in response
to IL-1 signaling including IL-6 (4). Ip injection of LPS into
wild-type mice produced no extracellular IL-1
and only small
quantities of IL-6. However, following challenge with extracellular
ATP, these LPS-primed mice generated cell-dissociated IL-1
and, in
turn, much greater levels of IL-6. In contrast, LPS-primed
P2X7R
/
animals failed to
generate IL-1 in response to ATP challenge and failed to match the
large increase in IL-6 production demonstrated by their wild-type
counterparts. These observations indicate that IL-1 released as a
result of the P2X7R can serve as a functional element of a
cytokine network. Although the ATP-dependent IL-6 response
of the knockout animals was greatly attenuated relative to the
wild-type, the knockout animals did appear to respond to ATP.
LPS-primed P2X7R
/
animals that
were subsequently challenged with PBS yielded no significant IL-6 at
4-h post-LPS priming. On the other hand, comparable animals challenged
with ATP yielded 6 ng/ml of IL-6 at the 4-h time point. This suggests
that ATP may work through other purinoceptors to affect IL-6
production. Perhaps ATP activation of G-protein-coupled P2Y-type
receptors leads to changes in intracellular cAMP and/or Ca2+ that enhance the LPS-induced IL-6 production response
(48). Additional work will be required to understand this
P2X7R-independent response, and studies are ongoing to
further characterize in vivo inflammatory processes within
the receptor-deficient mice to gain a more complete understanding of
how absence of this receptor affects immune system function.
IL-1 is a potent mediator of inflammatory responses (1). Not
surprisingly perhaps, animals appear to have developed a number of
safeguards to ensure that IL-1 activity is tightly regulated. For
example, a natural receptor antagonist, IL-1ra, exists and this protein
competes with IL-1 for binding to the type-1 IL-1 receptor (65). Unlike
IL-1
and IL-1
, IL-1ra binding does not lead to receptor
activation. Rather, the receptor antagonist appears to be produced via
an endogenous process to suppress IL-1 signaling events. Another
safeguard exists in the type-2 IL-1 receptor. Binding of IL-1 to this
receptor does not initiate signaling cascades within target cells (66).
Rather, the type-2 receptor appears to act as a decoy and provides a
mechanism to buffer and/or blunt an IL-1 biologic response. Finally,
the unique post-translational requirements of IL-1 appear to offer yet
an additional safeguard. Monocytes and macrophages may become activated
to produce the procytokine polypeptides, but these remain latent unless
the producing cells encounter a secondary stimulus that promotes their
post-translational processing and release. The
P2X7R
/
mice establish that ATP
acting through the P2X7R represents one potential mechanism
by which IL-1 post-translational processing is achieved in
vivo. Understanding how P2X7R activity is
regulated, therefore, may provide important insights for
designing strategies to control inflammatory response mechanisms.