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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 10 (November 15), 1998:
pp. 3521-3528
RAPID COMMUNICATION
Blockade of Human P2X7 Receptor Function With a Monoclonal
Antibody
By
G. Buell,
I.P. Chessell,
A.D. Michel,
G. Collo,
M. Salazzo,
S. Herren,
D. Gretener,
C. Grahames,
R. Kaur,
M.H. Kosco-Vilbois, and
P.P.A. Humphrey
From the Glaxo Institute for Molecular Biology, Geneva, Switzerland;
and the Glaxo Institute for Applied Pharmacology, Department of
Pharmacology, University of Cambridge, Cambridge, UK.
 |
ABSTRACT |
A monoclonal antibody (MoAb) specific for the human P2X7
receptor was generated in mice. As assessed by flow cytometry, the MoAb
labeled human blood-derived macrophage cells natively expressing P2X7 receptors and cells transfected with human
P2X7 but not other P2X receptor types. The MoAb was used to
immunoprecipitate the human P2X7 receptor protein, and in
immunohistochemical studies on human lymphoid tissue, P2X7
receptor labeling was observed within discrete areas of the marginal
zone of human tonsil sections. The antibody also acted as a selective
antagonist of human P2X7 receptors in several functional
studies. Thus, whole cell currents, elicited by the brief application
of 2 ,3-(4-benzoyl)-benzoyl-ATP in cells expressing human
P2X7, were reduced in amplitude by the presence of the
MoAb. Furthermore, preincubation of human monocytic THP-1 cells with
the MoAb antagonized the ability of P2X7 agonists to induce
the release of interleukin-1 .
© 1998 by The American Society of Hematology.
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INTRODUCTION |
P2X RECEPTORS ARE ligand-gated ion
channels that are activated by extracellular ATP. Their activation
results in the opening of a cationic channel with significant
permeability to calcium and intracellular
depolarization.1,2 In contrast to other P2X receptors
(P2X1-63), P2X7 is uniquely
bifunctional. When stimulated briefly by low concentrations of agonist,
the receptor acts as a nonselective cation channel.
However, repeated or prolonged application of higher agonist
concentrations, especially in solutions containing low concentrations
of extracellular divalent cations, creates a much larger aqueous pore.
Formation of this pore allows entry of fluorescent DNA binding dyes
such as YO-PRO-1 (629 Daltons) and eventually leads to cell lysis.
These same responses to ATP have been shown for native P2 receptors
expressed by mast cells, macrophages, or microglia and were previously
referred to as P2Z receptors.4 Among all ligand-gated ion
channels, P2X7 receptors thus have two remarkable features:
they appear only in the immune system, and there they can mediate
ATP-induced cell death.
Investigation of P2X7 receptors in the immune system has
suggested a potentially important role in immune responses. In either macrophages or microglial cells, P2X7 receptors are
functionally upregulated by lipopolysaccharide (LPS) or interferon-
(-IFN).5-7 Stimulation of P2X7 receptors
leads to the release of mature interleukin-1 (IL-1) in
macrophages8 and microglial cells9 and the
induction of phospholipase D activity as demonstrated in THP-1
cells.7 Recent evidence in microglial cells has shown an
unusual p65 homomeric form of NF B produced by P2X7
activation, suggesting a unique transcriptional activation
pathway.10 Finally, the extracellular ATP-induced killing
of mycobacteria in infected human macrophages has been shown to be
mediated by P2X7 receptors.11 Whether this killing of vesicle encapsulated bacteria is related to the cell fusion
observed in macrophage cultures expressing high levels of
P2X7 receptors is currently unknown.12
The functional study of P2X receptors has been hindered by the relative
absence of good subtype specific antagonists (see discussion). We
describe here a monoclonal antibody (MoAb) to human P2X7
that is both species and subtype specific and that has been found,
unexpectedly, to functionally antagonize the activation of both
recombinant and endogenous P2X7 receptors by extracellular ATP.
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MATERIALS AND METHODS |
MoAb generation and flow cytometry.
Human P2X72 was expressed in a Balb/c mouse
myeloma cell line, XS63 (ATCC TIB-17), by stable transfection. Balb/c
mice were immunized on days 0, 7, and 28 subcutaneously in the limbs and behind the neck with 107 transfected cells per
injection in MPL+TDM emulsion (RIBI; Inotech, Dottikon, Switzerland).
Three days after the final injection, the draining lymph nodes were
obtained and the tissue was digested using a DNase and collagenase
cocktail as reported elsewhere.13 The resulting cell
suspension was resuspended at 106 cells/mL and fused with
Sp2 myeloma cells using a standard protocol.14 The
hybridomas were selected in HAT medium, and 7 to 10 days after fusion,
the supernatants were harvested for differential screening by flow
cytometry on the transfected and nontransfected XS63 cells. Briefly,
cells were washed with FACS buffer (1% bovine serum albumin [BSA]
and 0.01% Na Azide in phosphate-buffered saline [PBS]) and successively incubated for 30 minutes with 50 µL supernatant, followed by washing, and a fluorescein isothiocyanate (FITC)-labeled sheep antimouse F(ab)2 fragment (Silenius
Laboratories, Hawthorn, Australia) diluted 1/100 in FACS buffer. Mean
fluorescence intensity was measured using a FACSCalibur (Becton
Dickinson, Erembodeggen, Belgium). A similar method was used to
investigate the effects of the antibody on HEK-293 cells transfected
with human P2X115 or human
P2X4.16 Antibodies were purified by
chromatography on Protein A Sepharose Fast Flow in PBS and eluted in
0.1 mol/L citrate, pH 4.5. Eluates were then subjected to gel
filtration on Superdex-200 (Pharmacia, Uppsala, Sweden) equilibrated in
PBS.
In some experiments, FITC-labeled MoAb was used to investigate the
specificity of the MoAb. Briefly, wild-type HEK293 cells (5 × 105 cells per well) or HEK293 cells expressing
hP2X3 or hP2X7 receptors were incubated with
the FITC-labeled MoAb for 6 hours in PBS containing 1% BSA. After 3 washes in PBS, cell-associated fluorescence was measured using
spectrofluorimeter. Specific fluorescence signals were obtained by
subtraction of relative fluorescence units (RFU) obtained in wild-type
cells from those determined from the transfected cells.
Preparation of human monocytes.
Human monocytes were isolated by one-step Ficoll gradient
separation6 and selected by forward and side scatter
profiles by flow cytometry (FACSVantage; Becton Dickinson). The
purified monocytes were then stimulated for 1, 2, and 3 days with LPS
(1 µg/mL) or -IFN (10 ng/mL).
Immunoprecipitation of human P2X7 receptors.
XS63 cells (5 × 106), transfected with
hP2X7 or vector alone, were resuspended in PBS at 4°C
and 40 µL biotinylation reagent (Amersham, Buckingham, UK) was added
for 20 minutes with mixing. Cells were washed three times with PBS and
lysed by the addition of cold extraction buffer (1% Triton X-100, 20 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1 mmol/L
CaCl2, and 1 mmol/L MgCl2) in the presence of
protease inhibitors (4 µmol/L phenylmethylsulfonyl fluoride, 2 µg/mL pepstatin, 2 µg/mL leupeptin, 2 µg/mL trypsin inhibitor,
and 2 µg/mL aprotinin; Sigma, St Louis, MO). After 20 minutes, the
extract was centrifuged at 14,000 rpm for 10 minutes at 4°C. The
resulting supernatant was incubated with 6 µg MoAb plus a mixture of
protein A and protein G beads (Pharmacia, Uppsala, Sweden) on a roller
mixer for 16 hours at 4°C. The beads were recovered by
centrifugation and washed with extraction buffer, and the immune
complexes were eluted by boiling for 2 minutes in Laemmli sample
buffer.17 Biotinylated proteins were visualized by 8%
sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis under
reducing conditions, transfer to nitrocellulose membranes, incubation
with peroxidase-coupled streptavidin, and development with the ECL
system (Amersham).
Immunohistochemistry on human tonsil.
Human tonsils were embedded in Tissue-Tec (Miles Inc, Naperville,
IL) and frozen on dry ice. Seven-micron to 10-µm thick
sections were air-dried for 1 hour and then fixed in acetone for 10 minutes before storage at  70°C. For immunohistochemistry,
the sections were incubated with 10 µg/mL primary antibody for 30 minutes at room temperature (RT), followed by incubation with
biotin-conjugated rat antimouse IgG-specific F(ab)2
fragments (Jackson Immunoresearch Laboratories, West Grove,
PA) for 30 minutes at RT. The labeling was shown using the
ABC kit and diaminobenzidine substrate (as described in the
manufacturer's protocol; Vector Laboratories, Burlingame,
CA). The primary antibodies were either mouse antihuman CD3 (Leu-4), mouse antihuman CD20 (Leu 16; both purchased from Becton
Dickinson), or the anti-P2X7 MoAb. The sections were
counterstained with May-Grunwald-Giemsa.
Electrophysiological recording.
Whole cell recordings18 were made to investigate the
effects of the MoAb on nucleotide-evoked inward currents from a variety of P2X receptor subtypes. For all experiments, HEK293 cells transfected stably with the indicated receptors were used. Recordings were made
essentially as described elsewhere.19 Briefly, cells were perfused with either a normal (consisting of 145 mmol/L NaCl, 2 mmol/L
KCl, 2 mmol/L CaCl2, 1 mmol/L
MgCl2, 10 mmol/L HEPES, 10 mmol/L D-glucose, pH 7.3;
osmolarity, 300 mOsm) or low-divalent cation containing (as described
above, but with only 0.5 mmol/L Ca2+ and without added
Mg2+) solution.
Agonists were applied using a computer-controlled fast-flow U-tube
system20 modified to include an extra solenoid valve. The
following agonists were used to evoke inward currents:
2,3-(4-benzoyl)-benzoyl-ATP (BzATP; human, rat, and mouse
P2X71,2,19) at 300 µmol/L, ATP (human
P2X421) at 10 µmol/L, and
 ,-methylene-ATP (meATP; human
P2X316) at 3 µmol/L. Nucleotides were
obtained from Sigma (Poole, UK). All experiments were performed at room
temperature (22°C to 24°C).
Measurement of BzATP-stimulated IL-1 release from THP-1 cells.
THP-1 cells (ECAC, Porton Down, UK) were grown in a humidified
atmosphere (95% air 5% CO2) at 37°C as a suspension
culture in RPMI 1640 with 10% heat-inactivated fetal bovine serum
(FBS; GIBCO, Paisley, UK). To measure release of IL-1, cells were
resuspended at 1 × 106 cells/mL in fresh
media containing 10 µg/mL LPS for 18 hours at 37°C. Cells were
centrifuged at 200g for 5 minutes and resuspended in assay
buffer composed of 140 mmol/L NaCl, 5 mmol/L KCl, 10 mmol/L glucose, 1 mmol/L CaCl2, 10 mmol/L HEPES, and 10 mmol/L N-methyl-D-glucamine and supplemented with 0.1% BSA (pH 7.4 at 37°C). After a second centrifugation as described above, the cells were resuspended in assay buffer at 37°C and 100 µL of cell
suspension was added to the wells of a 96-well V-bottom plate (150,000 cells per well) containing 100 µL of antibody. After preincubation
for 30 minutes at 37°C, BzATP or ATP was added and incubations were continued for 30 minutes. The plates were subsequently centrifuged at
200g for 5 minutes and 10 µL aliquots of the supernatants
were removed for determination of IL-1 release using a reporter
bioassay.22 In this assay, supernatants from the THP-1
cells were added to an A549 cell line that expresses the human IL-1
receptor and that has been genetically modified to secrete soluble
placental alkaline phosphatase (SPAP) in response to IL-1. These
cells were the generous gift of Dr Keith Ray (Glaxo Wellcome,
Stevenage, UK). The A549 cells were cultured in Dulbecco's modified
Eagle's medium containing 10% FBS at 37°C in a humidified
atmosphere (95% air, 5% CO2) for 16 hours, during which
time the cells released SPAP. To quantify the SPAP release evoked by
IL-1, aliquots of the A549 cell supernatants were transferred to
fresh plates and heated at 60°C for 30 minutes to inactivate
nonspecific phosphatase activity. After cooling to 22°C, 200 µL
of 5 mmol/L para-nitrophenol phosphate (pNpp) substrate, dissolved in 1 mol/L diethanolamine, 0.28 mol/L NaCl, and 0.5 mmol/L MgCl2
(pH adjusted to 9.85 with HCl), was added to the wells. SPAP activity
was determined by absorbance at 405 nm with time. The release of SPAP
from A549 cells was proportional to the concentration of IL-1
applied to the cells, and the IL-1 released from the THP-1 cells was
determined by a calibration curve based on human recombinant IL-1
(R&D Systems, Minneapolis, MN). The reporter assay was validated by
showing that 0.2 µg/mL of a neutralizing MoAb against human IL-1
(R&D Systems) was able to eliminate the effect of both human
recombinant IL-1 and the THP-1 cell supernatants on the release of
SPAP from A549 cells (data not shown).
| |
RESULTS |
The human P2X7 receptor was expressed in XS63 cells, a
Balb/c myeloma. These stably transfected cells reacted with a
previously characterized low-titer rabbit polyclonal antisera generated
to the C-terminal peptide of rat P2X7.23
Application of BzATP, a selective agonist for P2X7
receptors, resulted in cell swelling and permeability to the propidium
dye, YOPRO-1 (data not shown). HEK293 cells transfected with either the
rat or human P2X7 receptor cDNAs displayed the same
properties.1,2
Hybridomas were generated with lymph node cells from Balb/c mice
immunized with the human P2X7 receptor-expressing cells. Screening of the hybridoma supernatants by FACS analysis on the transfected XS63 cells yielded an IgG2b MoAb that reacted strongly with
cells expressing human P2X7 receptor
(Fig 1). The MoAb bound to the surface of
HEK293 cells bearing hP2X7 receptors (Fig 1) but not to
HEK293 cells that expressed human P2X1 or human
P2X4 (Fig 1A and B). In studies using the FITC-labeled MoAb
for hP2X7 transfected HEK293 cells, the cell-associated
specific fluorescence approached saturation at an MoAb concentration of
1 µg/mL (KD = 58 ± 18 ng/mL;
Bmax = 1,349 ± 12 specific RFU). In contrast, there was
no detectable specific binding of the MoAb binding to hP2X3
transfected HEK293 cells (the RFU value of 101 ± 19 at 1 µg/mL of
the MoAb was not significantly different from the value of 130 ± 10 RFU determined in wild-type HEK293 cells).
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| Fig 1.
Characterization of hP2X7 receptor MoAb by
flow cytometry with HEK293 cells stably transformed with (A)
hP2X1, (B) hP2X4, or (C) hP2X7.
Cells detached with PBS plus 1 mmol/L EDTA were incubated on ice with
15 µg/mL purified antibody for 30 minutes. The MoAb (bold line) was
detected with an FITC-labeled sheep antimouse F(ab)2
fragment. An IgG2b antibody (thin line) was used as an isotype
control.
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The MoAb also recognized the native P2X7 receptor on human
monocytes and macrophages
(Fig 2). Previous work had
functionally demonstrated P2X7 receptors on this cell type
and shown an enhanced activity during the monocyte to macrophage
transition.5,6 Human monocytes were derived from peripheral
blood mononuclear cells and cultured for 1, 2, or 3 days in media alone
or in media supplemented with LPS or -IFN. Compared with the isotype
control, the MoAb to hP2X7 receptor showed significant
reactivity with cells cultured in media alone at all three time
periods. This reactivity was enhanced by addition of either LPS or
-IFN. Interestingly, whereas P2X7 expression was
augmented within 24 hours of -IFN treatment and continued to be
high, LPS showed a more marginal upregulation, needing 48 hours for
augmentation (Fig 2A and B).
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| Fig 2.
Detection of P2X7 receptors by flow
cytometry on human blood-derived monocytes cultured for (A) 1 day, (B)
2 days, or (C) 3 days in complete RPMI medium (monocytes) or with the
addition of LPS (10 µg/mL) or -IFN (10 ng/mL). Antibodies were
used as in Fig 1, with the control showing incubation of monocytes with
an IgG2b isotype control. Monocytes were derived from PBMC by rosetting
and FACS using forward and side scatter.
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Next, the ability of the MoAb to immunoprecipitate the human
P2X7 receptor protein was tested. XS63 cells transfected
with human P2X7 receptor cDNA or mock transfected (vector
alone) were surface labeled by biotinylation and lysed to generate
membrane protein extracts. These were subjected to immunoprecipitation with the MoAb, followed by polyacrylamide gel electrophoresis (PAGE)
analysis (Fig 3). As detected by labeled
streptavidin, the MoAb was able to immunoprecipitate a major protein of
approximately 72 kD from the hP2X7 receptor containing
extract, but not from mock-transfected cells. Minor bands of 74 and 56 kD were also visible.
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| Fig 3.
Immunoprecipitation of hP2X7 receptor from
stably transfected XS63 cells with MoAb. Biotinylated surface proteins
from XS63 cells, transfected with either human P2X7 cDNA or
vector alone (control lane), were immunoprecipitated with the antihuman
P2X7 MoAb. The band was visualized using a streptavidin-peroxidase
conjugate. Markers at left are in kilodaltons of protein.
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Using serial cryostat sections of tonsils, the expression of
P2X7 in human lymphoid tissue was evaluated
(Fig 4). Numerous distinct cells (Fig 4C
and D) with dendritic morphology (Fig 4D, insert) labeled strongly in
the area of the marginal zone. In addition, a more diffuse lighter
labeling was detected within the light zone of the germinal center (GC,
Fig 4C) and in the marginal zone. These observations suggest that
macrophages as well as certain dendritic cells express P2X7
within tonsils.
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| Fig 4.
Expression of P2X7 receptor in human tonsil
by immunohistochemistry. Serial cryostat sections were processed to
show the location of P2X7 receptors using the
anti-P2X7 receptor MoAb. (A and B) Irrelevant isotype
(IgG2b) control MoAb; (C and D) anti-P2X7 MoAb; (E and G)
anti-CD3 (showing T cells) and anti-CD20 (showing B cells). (B), (D),
(F), and (H) are higher magnifications of (A), (C), (E) and (G),
respectively. GC, germinal center; MZ, marginal zone; T, T-cell zone.
Inset in (D) shows a higher magnification of one of the positive cells
in the marginal zone to detail the morphology.
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Inward currents evoked by BzATP in HEK293 cells transfected with human
P2X7 receptor were inhibited by incubation of the cells with the MoAb. This inhibition was concentration-dependent, and currents were reduced to approximately half maximal with 200 ng/mL antibody (Fig 5). Assuming a molecular
weight of 150 kD, the estimated IC50 value for the antibody
was about 5 nmol/L. The effects of the antibody were highly specific
for the human P2X7 receptor; currents evoked in HEK293
cells transfected with the mouse or rat orthologues of the
hP2X7 receptor or in cells transfected with
hP2X421 or hP2X316 were
unaffected by the MoAb (Fig 5) applied at concentrations that caused
greater than 80% inhibition of the currents observed in the human
P2X7-expressing cells. Application of the antibody alone to
human P2X7-expressing cells produced no inward currents.
Blockade of the human P2X7 receptor by the MoAb was only
slowly reversible, such that after 30 minutes of washing,
agonist-evoked inward currents were still inhibited by approximately
70% of their control values (Fig 5A).
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| Fig 5.
Inhibition of nucleotide-induced currents in HEK293 cells
stably transfected with hP2X7 by MoAb. (A) Comparison of
effects on various P2X receptors. In each panel, an initial application
of an appropriate purinergic agonist (see Materials and Methods) was
made to HEK293 cells stably transfected with one of five P2X receptors
(line 1). Cells were incubated with the MoAb (1.15 µg/mL) for 10 minutes and a second application of the same agonist was made in the
presence of the MoAb (line 2). A final application of the agonist was
made after 10 minutes of washing (except for hP2X7, which
was for 30 minutes) in the absence of the MoAb (line 3). (B)
Concentration-dependent inhibition of hP2X7 channel
function by the MoAb. Points represent the percentage of maximal
current after incubating cells for 10 minutes in varying concentrations
of the MoAb.
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BzATP or ATP evoked a concentration- and time-dependent release of
IL-1 from LPS-treated human monocytic cells (THP-1). The ability of
both -IFN and LPS to induce P2X7 receptor activity in
THP-1 cells has been previously shown.7 IL-1 release,
measured by bioassay (see Materials and Methods), increased more that
30-fold with maximum agonist stimulation (512 µmol/L BzATP for 30 minutes; 16.0 ± 1.0 ng IL-1 per 150,000 cells was released
v 0.4 ± 0.05 ng from control cells). Incubation of THP-1
cells with the MoAb caused a concentration-dependent inhibition of
IL-1 release, such that significant inhibition of the BzATP-induced
release could be obtained with the MoAb at a concentration of 38.3 ng/mL (Fig 6).
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| Fig 6.
Inhibition of BzATP-stimulated IL-1 release from THP-1
cells by hP2X7 receptor MoAb. THP-1 cells, pretreated with
LPS for 18 hours, were incubated (150,000 per well) at 37°C for 30 minutes in the absence ( ) or presence of ( ) 0.012 µg/mL, ( )
0.038 µg/mL, ( ) 0.12 µg/mL, ( ) 0.38 µg/mL, or ( ) 1.2 µg/mL of the hP2X7 MoAb. BzATP was added and, after 30 minutes of incubation at 37°C, the cell suspension was centrifuged
at 200g for 5 minutes and the IL1- present in a 10-µL
aliquot of supernatant was determined using a reporter assay as
described in the Materials and Methods. The data are the mean ± standard error of the mean of three experiments. In each experiment,
the maximal release of IL-1 was determined and the data were
expressed as a percentage of this release (0% and 100% represent 0.4 ± 0.05 and 16 ± 1 ng of IL-1 per well, respectively).
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DISCUSSION |
The aim of this study was to obtain an antibody directed against the
external domain of the human P2X7 receptor. This was achieved by immunizing Balb/c mice with mouse cells expressing recombinant hP2X7 receptor to maximize the potential for
raising antibodies to the intact protein. The hybridomas obtained were then screened by differential flow cytometry using nonpermeabilized cells expressing the human P2X7 receptor to obtain an
antibody that recognized the external domains of the receptor.
The isolated antibody was highly selective for human P2X7
receptors and did not recognize human P2X1 and human
P2X4 receptors by flow cytometry. These receptors are the
only P2X receptors so far localized to immune cells. The MoAb also
failed to label or affect responses at the human P2X3
receptor, and functional data suggest that the MoAb does not recognize
rat or mouse P2X7 orthologues.
The antibody was found to be suitable for quantitative analysis of cell
surface receptor expression and could be used to detect P2X7 receptors in THP-1 cells and to confirm the results of
earlier functional studies that have suggested that the human
P2X7 receptor is upregulated by -IFN and to a lesser
extent by LPS.7 Functionally, P2X7 receptors
have been primarily localized to myeloid lineages and we noted by flow
cytometry that the MoAb reacted with another human myeloid cell line,
U937 (not shown). However, there have also been reports of
ATP-activated channels with similar operational characteristics to the
P2X7 receptor, but that apparently lack the ability to form
the large pore in Epstein-Barr virus (EBV)-transformed lymphoblasts24 and lymphocytes from chronic lymphocytic
leukemia lymphocytes,25 and this MoAb should provide a
useful tool for identification of P2X7 receptor-containing
assemblies. The finding that the antibody was also effective at
immunoprecipitating the P2X7 receptor from cells known to
express the receptor will also enable it to be used for the detection
of other subunits or proteins that may interact with the
P2X7 receptor.
Previous in situ hybridization studies have shown that rat
P2X7 receptor is most abundant in bone marrow; in the
brain, it has only been observed in microglial cells.23 The
localization of distinct cell populations within the tonsil is itself
an interesting finding and demonstrates the utility of this MoAb to
investigate P2X7 protein expression using
immunohistochemical methods. The MoAb labeled a cellular subpopulation
of the marginal zone and T-cell area that presented a distinct
morphology from lymphocytes (inset, Fig 4D). Because macrophages and
dendritic cells are found in these areas, respectively, and both can be
derived from a common myeloid precursor26 known to express
P2X7 receptors,27 we hypothesize that certain
antigen-presenting cells express the P2X7 receptor during
immune responses. Future studies will be aimed at defining these
populations and characterizing the functional significance of their
P2X7 receptor expression.
The MoAb was selected by flow cytometry for surface binding to
nonpermeabilized cells that express the hP2X7 receptor (Fig 1). The epitope recognized is presumably present in the extracellular loop of the native receptor, because the MoAb demonstrated functional antagonism (see below). However, it is likely that contributions to the
epitope are made from the tertiary or quaternary structures of the
receptor, because all attempts to use the MoAb in Western blot
experiments with denatured proteins failed, and yet immunoprecipitation of native protein was successful.
The study of ATP as an extracellular modulator in the immune system has
been largely hampered by lack of pharmacological tools. The commonly
used P2 receptor antagonists, such as PPADS and suramin, are relatively
weak at the hP2X7 receptor2 (but see Chessell et al28) and are unable to differentiate between P2X
receptor subtypes. In addition, many of these antagonists also show
affinity for P2Y receptors or have a highly nonspecific
profile.29 Oxidized ATP, an irreversible antagonist, has
been used to characterize P2X7 receptor responses, but
seems unlikely to be specific, because this compound binds to several
ATP-binding proteins.30 Furthermore, we have found in
functional studies that oxidized ATP is as effective at blocking the
rat P2X2 receptor as it is in blocking the
hP2X7 receptor (A.D. Michel, unpublished
observation). One of the major findings of this study is
the demonstration that the MoAb is an effective antagonist of
BzATP-induced P2X7 channel activation. The binding of the
MoAb to human P2X7 receptors occurred with relatively high
affinity, giving an estimated IC50 of 5 nmol/L to inhibit
inward currents, and was only slowly reversible. Because the MoAb did
not cross-react with P2X1 or P2X4 receptors and
was inactive as an antagonist of responses mediated by
P2X1, P2X3, or P2X4 receptors, it
represents a unique tool for the identification of putative
P2X7 receptors and for determining the potential coassembly of P2X7 with other P2X subunits. Considerable evidence,
both biochemical31 and functional,32 exists for
the heteromeric assembly of other P2X channels (P2X2 + P2X3) in the peripheral nervous system, and disparate
findings of the characteristics of P2Z receptors in differing systems
may be explained by heteropolymeric combination of P2X7
with other P2X subunits.
ATP-induced IL-1 release has been previously reported from activated
macrophage and microglial cells.9,33 Our observation of
inhibition of this release from the myeloid cell line, THP-1, by the
MoAb (Fig 6) is important, demonstrating unequivocally that this effect
of ATP is mediated by a native receptor (P2Z) containing
P2X7 subunits, even though THP-1 cells express other ATP
receptors, including P2U7 (now known as P2Y2)
receptors. Other phenomena distal to the activation of P2Z receptors,
such the induction of NF-B and caspase10 or the killing
of intracellular mycobacteria,11 can now be investigated by
functional antagonism using the MoAb.
The demonstration of functional receptor antagonism of a
neurotransmitter ligand with an MoAb is not unique and has been
previously described for the nicotinic receptor.34,35
However, in the absence of specific P2X7 antagonists, the
MoAb described in the present study represents a unique tool with which
to explore the function of the P2X7 receptor. Further
studies will be required to determine if the receptor blocking
activities of the antibody are a consequence of a direct interaction
with the ATP-binding site or accessory sites or are simply a
consequence of steric hindrance of either ligand-binding or channel
opening.
| |
FOOTNOTES |
Submitted July 2, 1998;
accepted August 20, 1998.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to I.P. Chessell, PhD, Glaxo
Institute for Applied Pharmacology, Department of Pharmacology,
University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, UK;
e-mail: ic44126{at}glaxowellcome.co.uk.
| |
REFERENCES |
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Surprenant A, Rassendren F, Kawashima E, North RA, Buell G:
The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7).
Science
272:735, 1996[Abstract]
2.
Rassendren F, Buell G, Virginio C, North RA, Surprenant A:
The permeabilizing ATP receptor (P2X7): Cloning and expression of a human cDNA.
J Biol Chem
272:5482, 1997[Abstract/Free Full Text]
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Abstract
Introduction
