Am J Physiol Cell Physiol
279: C1189–C1197, 2000.
Expression of P2X7 purinoceptors on human lymphocytes
and monocytes: evidence for nonfunctional P2X7 receptors
B. J. GU,1 W. Y. ZHANG,1 L. J. BENDALL,1 I. P. CHESSELL,2 G. N. BUELL,3 AND J. S. WILEY1
1
Department of Medicine, Nepean Hospital, University of Sydney, Penrith, New South Wales 2750,
Australia; 2Department of Pharmacology, University of Cambridge, Cambridge CB2 1QJ,
United Kingdom; and 3Department of Molecular Biology, Geneva Institute for
Biomedical Research, 1228 Geneva, Switzerland
Received 8 December 1999; accepted in final form 26 April 2000
Gu, B. J., W. Y. Zhang, L. J. Bendall, I. P. Chessell,
G. N. Buell, and J. S. Wiley. Expression of P2X7 purinoceptors on human lymphocytes and monocytes: evidence for
nonfunctional P2X7 receptors. Am J Physiol Cell Physiol 279:
C1189–C1197, 2000.—Lymphocytes from normal subjects
and patients with B-chronic lymphocytic leukemia (B-CLL)
show functional responses to extracellular ATP characteristic of the P2X7 receptor (previously termed P2Z). These
responses include opening of a cation-selective channel/pore
that allows entry of the fluorescent dye ethidium and activation of a membrane metalloprotease that sheds the adhesion
molecule L-selectin. The surface expression of P2X7 receptors
was measured in normal leucocytes, platelets, and B-CLL
lymphocytes and correlated with their functional responses.
Monocytes showed four- to fivefold greater expression of
P2X7 than B, T, and NK lymphocytes, whereas P2X7 expression on neutrophils and platelets was weak. All cell types
demonstrated abundant intracellular expression of this receptor. All 12 subjects with B-CLL expressed lymphocyte
P2X7 at about the same level as B lymphocytes from normal
subjects. P2X7 function, measured by ATP-induced uptake of
ethidium, correlated closely with surface expression of this
receptor in normal and B-CLL lymphocytes and monocytes
(n ⫽ 47, r ⫽ 0.70; P ⬍ 0.0001). However, in three patients the
ATP-induced uptake of ethidium into the malignant B lymphocytes was low or absent. The lack of P2X7 function in these B
lymphocytes was confirmed by the failure of ATP to induce
Ba2⫹ uptake into their lymphocytes. This lack of function of the
P2X7 receptor resulted in a failure of ATP-induced shedding of
L-selectin, an adhesion molecule that directs the recirculation
of lymphocytes from blood into the lymph node.
extracellular adenosine 5⬘-triphosphate; adenosine 5⬘triphosphate-induced ethidium uptake; monocyte P2X7; lymphocyte P2X7; B cell chronic lymphocytic leukemia
for extracellular ATP have been demonstrated in cells of the immune and hemopoietic system.
Thus monocytes, macrophages, neutrophils, and mast
cells express metabotropic purinoceptors named P2Y
receptors, the occupancy of which by ATP or UTP
causes activation of the phosphoinositide signaling cascade, formation of inositol 1,4,5-trisphosphate, and release of Ca2⫹ from intracellular stores. A second major
RECEPTORS
Address for reprint requests and other correspondence: J. S. Wiley,
Clinical Sciences Bldg., Nepean Hospital, Penrith, NSW 2750, Australia (E-mail: [email protected]).
http://www.ajpcell.org
class of receptor for extracellular ATP is the P2X receptors, at which ATP acts as a ligand that opens a
cation-selective channel (5, 10). The P2X receptors
have two transmembrane domains with intracellular
amino and carboxy termini and an oligomeric structure
in the plasma membrane based on trimeric or larger
complexes of identical subunits (32). The seven members of the P2X receptor family have extensive homology (30–40%) in their primary structure but differ
considerably in the length of their carboxy termini,
ranging from as short as 30 amino acids for P2X6 and
48 amino acids for P2X1 to as long as 240 amino acids
for P2X7 (41). Most members of the P2X receptor family
have been characterized and cloned from smooth muscle and central and peripheral neurons, where they
function in cell-cell communication. The latest cloned
member of the P2X family, the P2X7 receptor (previously termed P2Z), is unique in being strongly expressed in cells of hemopoietic origin with a rank order
of agonist potency [3⬘-O_(4 benzoylbenzoyl)-ATP ⬎
ATP] that differs from that of other P2X receptors (19,
42, 45). This P2X7 ionic channel opened by extracellular ATP shows strong selectivity for the divalent cations Ca2⫹ and Ba2⫹ over monovalent cations (28, 31).
After immediate (⬍1 s) channel opening, a second
permeability state develops that allows larger organic
cations to pass, a process termed “pore” formation
(33, 43, 48). This larger permeability state allows
permeation by ethidium⫹ cation (314 Da) or YoPro2⫹
(375 Da) but excludes passage of propidium2⫹ cation
(414 Da).
The distribution of the P2X7 receptor has been inferred by permeability and reverse transcription-polymerase chain reaction (RT-PCR) analysis and reported
on cultured monocyte/macrophages, dendritic cells,
mast cells, transformed fibroblasts, and lymphocytes
from patients with B-chronic lymphocytic leukemia
(B-CLL) (7, 22, 24, 36, 43, 47). A number of downstream effects have been documented to follow P2X7
receptor activation that can be considered as either
rapid (over minutes) or delayed (over hours) effects.
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FUNCTIONAL AND NONFUNCTIONAL P2X7 RECEPTORS IN LEUCOCYTES
Activation of the P2X7 receptor in B lymphocytes
causes immediate opening of a cation-selective channel
and also leads to shedding of the adhesion molecules
L-selectin and CD23 via activation of a membrane
metalloprotease(s) (23). Moreover, ATP also stimulates
the activity of both Ca2⫹-dependent and -independent
phospholipase D, and all these effects occur within
minutes of ATP exposure (12, 18). In contrast, ATP has
been shown to induce apoptotic death or cytolysis of
human macrophages or murine thymocytes only after a
delay of several hours (1, 35, 51). The cytolytic effect of
even brief P2X7 receptor activation has been proposed
to result from sustained activity of intracellular phospholipase D, the action of which generates a delayed
permeability lesion in the cell membrane (13). These
delayed effects show a time frame in keeping with
ATP-induced activation of the transcription factor nuclear factor-␬B (15), and there is strong evidence that
activation of P2X7 also stimulates the activity of intracellular caspases before ATP-induced apoptosis (14).
This stimulation of caspase activity may also be responsible for ATP-induced processing of pro-interleukin (IL)-1␤ to the cytokine IL-1␤ in activated macrophages (34).
This study of the human P2X7 receptor has taken
advantage of the recent development of a monoclonal
antibody that is both species and subtype specific and
is directed against an extracellular domain on the
molecule (2). This latter property makes it suitable for
flow cytometric analysis of P2X7 expression in leucocyte subpopulations, and P2X7 function also can be
measured from ethidium uptake by flow cytometry.
The results indicate that purinoceptors of the P2X7
class are strongly expressed on both normal and leukemic lymphocytes but that, in some patients, this
receptor is functionally inactive.
MATERIALS AND METHODS
Materials. ATP, 3⬘-O-(4 benzoylbenzoyl)-ATP (BzATP),
ethidium bromide, barium chloride, D-glucose, bovine serum
albumin (BSA), RPMI 1640 medium, and the FluoroTag
FITC conjugation kit were purchased from Sigma Chemical
(St. Louis, MO). Fura 2-acetoxymethyl ester and the Alexa
488 protein labeling kit were from Molecular Probes (Eugene,
OR). AffiGel 10 was from Bio-Rad (Hercules, CA). FicollHypaque (density 1.077) was obtained from Amersham Pharmacia (Uppsala, Sweden). Fluorescein isothiocyanate
(FITC)- and R-phycoerythrin (RPE)-conjugated negative control antibodies, mouse anti-human CD41, CD3, CD14, CD16
antibodies, and RPE-Cy5-conjugated mouse anti-human
CD19 antibody were from Dako (Carpinteria, CA). The FITClabeled mouse anti-human monoclonal antibody to L-selectin
(CD62L) was the DREG.55 clone (Bender MedSystems, Vienna, Austria).
Preparation of leukocytes and platelets. Peripheral blood
from 9 normal subjects and 12 B-CLL patients was collected
and diluted with an equal volume of RPMI 1640 medium.
Mononuclear cells were separated by density gradient centrifugation over Ficoll-Hypaque, washed once in RPMI 1640
medium, and then resuspended in HEPES-buffered NaCl
medium (145 mM NaCl, 5 mM KCl, 1 mM CaCl2, 10 mM
HEPES, 5 mM D-glucose, and 1 mg/ml BSA, pH 7.5). Immunofluorescent staining with CD14 antibody showed that
monocytes comprised 0.5–2.3% of the B-CLL population and
5–11% of the normal mononuclear cells. For RT-PCR analysis of mRNA for P2X7 in B-CLL, B lymphocytes were sorted
to high purity on a Becton Dickinson FACS Vantage flow
cytometer. Immunofluorescent staining (n ⫽ 6) showed that
⬎99.8% of cells were CD19⫹, CD41⫺ lymphocytes. Polymorphonuclear granulocytes were collected after centrifugation
over Ficoll-Hypaque, and erythrocytes were removed by hypotonic lysis in lysing buffer (0.7% NH4Cl, 5 mM K2CO3, and
0.1 mM EDTA, pH 7.0) for 10 min at 4°C. The neutrophils
were washed twice and resuspended in HEPES-buffered
NaCl medium. Platelet-rich plasma (PRP) was obtained from
normal subjects by low-speed centrifugation (150 g for 5 min)
of whole blood. PRP was then centrifuged again at 200 g for
10 min to remove small contaminating leukocytes. Immunofluorescent staining showed that 99.9% of cells were strongly
CD41 positive.
Antibody production and preparation. Mouse anti-human
P2X7 receptor monoclonal antibody was prepared from hybridoma L4 supernate by chromatography on protein A-Sepharose
Fast Flow as described previously (2). Either FITC or Alexa 488
protein labeling kits were used to conjugate the P2X7 antibody
according to the manufacturer’s instructions. The conjugated
antibody had 1.2 FITC per IgG and was stored at 0.64 mg/ml at
4°C. Anti-P2X7 antibody showed no binding to the surface or
cytoplasm of HEK-293 cells, a cell line that does not express this
receptor in subconfluent conditions.
Immunofluorescent staining for P2X7 receptor. Suspensions of leucocytes (50 ␮l of 1.0 ⫻ 107 cells/ml in HEPESbuffered NaCl medium) or PRP (100 ␮l of 1–2 ⫻ 108 cells/ml)
were incubated with fluorescence-conjugated anti-P2X7 antibody (60 ␮g/ml) at room temperature for 20 min and washed
once with PBS (0.15 M NaCl and 0.01 M phosphate, pH 7.2).
RPE-labeled anti-CD41, anti-CD14, anti-CD16, or anti-CD3
together with RPE-Cy5-labeled anti-CD19 monoclonal antibodies were used as the second or third color. Labeled cells
were then resuspended in 1 ml of PBS and analyzed on a
Becton Dickinson FACSCalibur flow cytometer (San Jose,
CA). The isotype control was a murine FITC-conjugated
IgG2b. For intracellular staining, cells were first labeled with
RPE-conjugated anti CD41, CD14, CD16, or CD3 plus CD19
and then fixed and permeabilized with the use of a Fix &
Perm kit (Cal Tag, Jarfalla, Sweden) according to the manufacturer’s instructions before they were labeled with the
FITC-conjugated anti-P2X7 antibody.
ATP-induced shedding of L-selectin. Aliquots of lymphocytes (1.0 ⫻ 106 cells/ml) were incubated for up to 30 min in
1 ml of HEPES-buffered KCl medium (10 mM HEPES, 150
mM KCl, 5 mM D-glucose, and 0.1% BSA, pH 7.5) at 37°C
with 0.1 mM BzATP or 1.0 mM ATP. The incubation was
stopped by the addition of an equal volume of cold isotonic
Mg2⫹ (20 mM MgCl, 145 mM NaCl, 5 mM KCl, and 10 mM
HEPES, pH 7.5). Cells were washed once and stained with
FITC-conjugated anti-L-selectin monoclonal antibody. The
mean channel fluorescence was collected in linear mode.
Ethidium cation influx measurement by time-resolved flow
cytometry. Mononuclear cells (2 ⫻ 106) prelabeled with fluorophore-conjugated anti-CD3, anti-CD14, anti-CD16, or anti-CD19 were washed once and resuspended in 1.0 ml of
HEPES-buffered KCl medium at 37°C. Cells were gated by
forward and side scatter and by cell type-specific antibodies.
Ethidium (25 ␮M) was added, followed 20 s later by the
addition of 1.0 mM ATP. Mononuclear cells were analyzed at
1,000 events/s by flow cytometry, and the linear mean channel fluorescence intensity for each gated subpopulation over
successive 5-s intervals was analyzed with the use of WinMDI software (Joseph Trotter, version 2.7) and plotted
against time (48).
FUNCTIONAL AND NONFUNCTIONAL P2X7 RECEPTORS IN LEUCOCYTES
Cytosolic Ba2⫹ measurements by fluorometry. Lymphocytes (1 ⫻ 107 cells/ml) were washed once and loaded with 2
␮M fura 2-acetoxymethyl ester by incubation at 37°C for 30
min in Ca2⫹-free HEPES-buffered NaCl medium. Cells were
washed once and left in HEPES-buffered NaCl with 1 mM
Ca2⫹ for another 30 min. Lymphocytes were then washed
twice and resuspended in 3 ml of HEPES-buffered KCl medium at a concentration of 2 ⫻ 106 cells/ml. These samples
were stirred at 37°C and stimulated with 1 mM ATP after the
addition of 1.0 mM BaCl2. Entry of Ba2⫹ into cells loaded
with fura 2 produces changes almost identical to those produced by Ca2⫹ in the excitation and emission spectra of fura
2 (37, 50). Fluorescence signals were recorded by a Johnson
Foundation fluorometer with excitation at 340 nm and emission at 500 nm. Calibration of maximal and minimal fluorescence intensities was performed after each run by the addition of 25 ␮M digitonin followed by 50 mM EGTA. Control
experiments showed that addition of ATP did not release
Ca2⫹ from the internal stores of lymphocytes suspended in
medium containing EGTA.
RT-PCR analysis for P2X7. Total RNA was isolated from
cells using an RNA isolation reagent (Advanced Biotechnologies, Epsom, UK) according to the manufacturer’s recommendations. For complementary DNA (cDNA) synthesis, 1
␮g of total RNA was used in the RT reaction with 0.5 ␮g of
oligo(dT) primer, heated for 10 min at 70°C, and then 1⫻
PCR buffer, 2.5 mM MgCl2, 0.5 mM dNTP (dATP, dGTP,
dCTP and dTTP) mix, and 0.01 M dithiothreitol were added
and incubated at 42°C for 5 min. Two hundred units of
C1191
SuperScript II reverse transcriptase (Life Technologies) were
added, and the tube was incubated at 42°C for 50 min. The
reaction was heated to 70°C to inactivate the reverse transcriptase. Finally, 1 ␮l of RNase A was added, and the tube
was incubated for 20 min at 37°C. To amplify the COOHterminal fragment of P2X7 receptor, we used the forward
primer 5⬘-TCTGCAAGATGTCAAGGGC-3⬘ (nucleotide position 1,286–1,304 in exon 12) and the reverse primer 5⬘TCACTCTTCGGAAACTCTTTCC-3⬘ (nucleotide position
1,780–1,759 in exon 13).
For performing PCR, a 25-␮l reaction contained 1⫻ PCR
buffer, 1.5 mM MgCl2, 100 ␮M dNTP (dATP, dGTP, dCTP,
and dTTP), 10 pmol each of P2X7 primer and 0.75 U Taq
DNA polymerase (Promega), and 100 ng of cDNA sample.
The PCR was carried out with a thermocycling program as
follows: initial denaturation at 95°C for 5 min and then 35
cycles of 95°C for 45 s, 52°C for 45 s, and 72°C for 1 min; the
final extension step was performed at 72°C for 10 min.
Detection of PCR products was performed by electrophoresis
in a 2% agarose gel and ethidium bromide staining. The size
of the PCR fragments was determined with a 100-bp DNA
molecular weight ladder and the nature of the products was
confirmed by DNA sequencing.
RESULTS
Surface expression of P2X7 on normal lymphocytes
and monocytes. In Fig. 1, the surface expression of
Fig. 1. Fluorescence histograms of P2X7 receptor expression on the surface (A) or after permeabilization (B) of
normal peripheral blood mononuclear cell subsets, granulocytes, or platelets. Cells were initially gated by forward
and side scatter and then by cell type-specific antibodies for monocytes (CD14⫹, CD19⫺), B lymphocytes (CD19⫹
and CD3⫺), T lymphocytes (CD3⫹ and CD19⫺), NK lymphocytes (CD16⫹, CD19⫺), neutrophils (CD41⫺), and
platelets (CD41⫹). With the exception of platelets, CD41-positive cells were excluded from the analysis. Shaded
line, isotype control; solid line, P2X7 antibody.
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FUNCTIONAL AND NONFUNCTIONAL P2X7 RECEPTORS IN LEUCOCYTES
Table 1. Expression and function of P2X7 receptor in
mononuclear cells from normal and B-CLL subjects
Relative P2X7
Expression, mean
channels of
fluorescence
intensity
58.5 ⫾ 16.2
13.1 ⫾ 2.4
5.6 ⫾ 1.6
9.7 ⫾ 2.7
n
Relative ATP-Induced
Ethidium Uptake,
arbitrary units
of area
n
8
9
9
8
18.9 ⫾ 1.8
3.5 ⫾ 0.6
4.0 ⫾ 1.1
4.3 ⫾ 0.5
5
6
6
6
23.2 ⫾ 2.0
7.4 ⫾ 1.1
3.4 ⫾ 8.9
6.9 ⫾ 1.0
6
6
6
6
Normal Subjects
Monocytes
B lymphocytes
T lymphocytes
NK cells
B-CLL Subjects (With Functional P2X7)
Monocytes
B lymphocytes
T lymphocytes
NK cells
69.9 ⫾ 15.0
21.7 ⫾ 4.3
9.3 ⫾ 1.7
19.6 ⫾ 2.7
9
9
9
8
Values are means ⫾ SE; n is no. of subjects, each studied once.
Mononuclear cell preparations (1 ⫻ 107 cells/ml) from normal and
B-chronic lymphocytic leukemia (B-CLL) subjects were incubated for
20 min at 20°C with fluorescein-conjugated P2X7 antibody plus
R-phycoerythrin-labeled surface marker antibody in HEPES-buffered saline containing 10% group AB serum. Cells were washed once
and analyzed for P2X7 expression on subpopulations gated for monocytes (CD14⫹, CD19⫺), B lymphocytes (CD19⫹, CD3⫺), T lymphocytes (CD3⫹, CD19⫺), and NK cells (CD16⫹, CD19⫺). Isotype control
values ranged from 2 to 8 mean channels of fluorescence intensity
and were subtracted from the values for each type. Function of P2X7
was measured as the area under the ethidium uptake curve, measured between 0 and 5 min of incubation with ATP (1.0 mM) at 37°C.
There was negligible uptake of ethidium in the absence of ATP.
P2X7 was measured by flow cytometry of normal white
cells stained with directly conjugated P2X7 antibodies.
Monocytes showed the highest expression of surface
P2X7 receptors, which was four- to fivefold higher than
P2X7 values for normal lymphocytes (Table 1, P ⬍
0.001). Lymphocytes of B, T, and NK cell lineage
showed lower and similar expression. Polymorphonuclear neutrophils showed weak expression for P2X7
when gated to exclude those cells that expressed CD41
as a result of platelet adhesion. Platelets were also
weakly positive for P2X7, whereas erythrocytes (data
not shown) were negative. As previously shown (2), the
cell line HEK-293 was also negative for surface P2X7
expression.
Intracellular expression of P2X7 on leucocytes and
platelets. Large amounts of P2X7 protein were found in
an intracellular location in monocytes, lymphocytes of
all subtypes, neutrophils, and platelets (Fig. 1B). Precise comparison of intracellular to cell surface P2X7
expression was not possible because of varying values
of the isotype (IgG2b) control. However, Fig. 1 clearly
shows that intracellular P2X7 expression is approximately an order of magnitude greater than expression
on the cell surface. The cell line HEK-293 showed no
intracellular staining with the P2X7 antibody (data not
shown).
P2X7 functional responses in normal monocytes and
lymphocyte subsets. ATP-induced uptake of ethidium
into mononuclear preparations from six normal subjects was measured by flow cytometry (48). Monocytes
and lymphocyte subsets were identified by using FITC-
or RPE-Cy5-conjugated antibodies that allowed the
relative ethidium uptakes to be compared among different cell types. Figure 2 shows uptake curves for
monocytes, NK, B, and T lymphocytes from one normal
subject and confirms that ethidium permeates via the
P2X7 pathway given that uptake by lymphocytes was
almost abolished in the presence of the P2X7 monoclonal antibody. When the area under the uptake curve
was used as an estimate of P2X7 permeability for six
normal subjects, there were no differences among these
three lymphocyte subsets (Table 1). However, ethidium
uptake through the P2X7 channel/pore was fivefold
greater for monocytes than for B lymphocytes of normal origin (Table 1, P ⬍ 0.001). ATP-induced ethidium
uptake into resting neutrophils and platelets was negligible (data not shown).
Surface and intracellular P2X7 in B-CLL lymphocytes. Our previous work showed that ATP-induced
ethidium uptake is mediated by the P2X7 receptor in
B-CLL lymphocytes (46). Fluorescence histograms of
the P2X7 antibody binding shows that B-CLL lymphocytes express P2X7 on their surface at about the same
level as observed for normal B lymphocytes (Fig. 3A
and Table 1). Moreover, intracellular levels of P2X7
were far higher than those found on the cell surface
(Fig. 3B) by approximately an order of magnitude. The
expression of P2X7 receptors on monocytes from the
B-CLL patients was fourfold greater than that on BCLL lymphocytes (Table 1, P ⬍ 0.001).
Functional and nonfunctional P2X7 channels. As
previously reported (46), lymphocytes from most patients with B-CLL showed strong ATP-induced uptake
of both Ba2⫹ and ethidium cation (Table 1 and Fig. 4A).
However, B lymphocytes from three patients failed to
show these ATP-induced permeability responses. Representative data are shown in Fig. 4A from subjects
Fig. 2. ATP-induced ethidium uptake into normal peripheral blood
mononuclear cell subsets. Mononuclear preparations (2 ⫻ 106 cells/
ml) prelabeled with cell type-specific antibodies were suspended in
HEPES-buffered isotonic KCl medium at 37°C. Ethidium was added,
followed 20 s later by the addition of 1.0 mM ATP (arrow). Mean
channel of cell-associated fluorescence intensity was measured for
each gated population at 5-s intervals. Addition of the P2X7 monoclonal antibody (MAb) greatly reduced the ethidium uptake into B
lymphocytes from normal subjects.
FUNCTIONAL AND NONFUNCTIONAL P2X7 RECEPTORS IN LEUCOCYTES
C1193
Fig. 3. Fluorescence histograms of P2X7 receptor expression on the surface (A) or after permeabilization (B) of
lymphocytes from 4 patients (CLL 1–4) with B-chronic lymphocytic leukemia (B-CLL). Lymphocytes prelabeled
with anti-CD19 antibody were analyzed before and after permeabilization to allow intracellular access of
FITC-P2X7 antibody. Shaded line, isotype control; solid line, P2X7 antibody.
CLL 1 and CLL 2, whose lymphocytes showed strong
ATP-induced ethidium uptake, but this response was
absent in B-CLL lymphocytes from subjects CLL 3 and
CLL 4. Similarly, lymphocytes from subjects CLL 1 and
CLL 2 showed strong ATP-induced uptake of Ba2⫹,
whereas lymphocytes from subjects CLL 3 and CLL 4
showed only minimal ATP-induced uptake of Ba2⫹ in
K⫹ medium (Fig. 4B). The function of the P2X7 receptor was also studied in monocytes from the B-CLL
subjects, because most studies of P2X7 function have
employed cells of monocytic origin. Figure 4C shows
that ATP-induced ethidium uptake into monocytes
from subjects CLL 3 and CLL 4 was below the range for
monocytes from either normal subjects or subjects CLL
1 or CLL 2. Recent data have suggested that removal of
extracellular Cl⫺ as well as extracellular Na⫹ enhances permeability responses and stimulates the
function of the P2X7 receptor (30). Thus ethidium uptake into B lymphocytes was measured in a buffered
sucrose medium with and without addition of BzATP,
the complete and most potent agonist for the P2X7
receptor (19). BzATP-induced ethidium cation influx
was greater in this Na⫹-free, low-Cl⫺ medium than in
KCl medium used in Fig. 4A (data not shown). However, Fig. 4D shows that the difference between functional (CLL-2) and nonfunctional (CLL-4) responses
was still evident in the sucrose medium.
Correlation of surface P2X7 expression and ATPinduced ethidium uptake. The expression of P2X7 receptors on the surface of normal leucocyte subsets and
on B lymphocytes and monocytes from P2X7 functional
B-CLL patients was compared with the ATP-induced
uptake of ethidium (Table 1). A close correlation was
found between receptor expression and function judged
by ethidium uptake (n ⫽ 47, r ⫽ 0.70; P ⬍ 0.0001).
Molecular analyses of P2X7 transcripts. It has been
previously reported that truncation of the long COOHterminal tail of the P2X7 receptor abolishes ATP-induced uptake of large fluorescent dyes such as YoPro2⫹
or ethidium cation (42). The presence of mRNA transcripts that included the COOH-terminal tail of P2X7
was examined in B-CLL lymphocytes that were sorted
to high purity to achieve ⬎99.8% cell purity. Lymphocytes from six patients (subjects CLL 1–CLL 5 and
subject CLL 8) showed an RT-PCR product of identical
size using primers specific for the long COOH-terminal
tail. The PCR products migrated at the 495-bp predicted size for the P2X7 product (Fig. 5). mRNA transcripts for the P2X7 tail were uniformly present on
B-CLL lymphocytes in all six patients examined, and
there were no gross differences among patients with
functional (subjects CLL 1, CLL 2, CLL 5, and CLL 8)
and nonfunctional P2X7 receptors (subjects CLL 3 and
CLL 4) (Fig. 5).
Failure of L-selectin shedding with nonfunctional
P2X7. Our previous results (25) showed that both ATP
and BzATP induce shedding of L-selectin via activation
of P2Z/P2X7 receptors. Figure 6 shows that BzATP, a
more potent agonist for the P2X7 receptor, caused
rapid and complete shedding of L-selectin within several minutes from the surface of B-CLL lymphocytes
with functionally active P2X7 receptors. In contrast,
BzATP produced very little shedding of L-selection
from lymphocytes with functionally inactive P2X7 receptors. Similar results were obtained when ATP was
used as agonist for the P2X7 receptor.
DISCUSSION
Much of the characterization of the P2X7 receptor
(previously termed P2Z) has been in cells of hemopoi-
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FUNCTIONAL AND NONFUNCTIONAL P2X7 RECEPTORS IN LEUCOCYTES
Fig. 4. A: ATP-induced ethidium uptake into B-CLL lymphocytes from 4 patients with B-CLL. Ethidium was added,
followed 20 s later by the addition of 1.0 mM ATP (arrow). Shaded area indicates the range of ethidium uptake observed
for B lymphocyte subpopulations from normal subjects. Mean channel of cell-associated fluorescence intensity was
measured for each B cell gated population at 5-s intervals. B: Ba2⫹ influx into normal and B-CLL lymphocytes.
Lymphocytes (6 ⫻ 106) loaded with 2 ␮M fura 2-AM were resuspended in 3 ml of HEPES-buffered KCl medium. Ba2⫹
(1.0 mM) was added 40 s before stimulation with 1.0 mM ATP (as indicated by arrows). Addition of the P2X7 monoclonal
antibody (P2X7-MAb) greatly reduced the Ba2⫹ influx into the normal lymphocytes. C: ATP-induced ethidium uptake
in monocytes from the same 4 patients shown in Fig. 3. Cells (2 ⫻ 106 cells/ml) were first marked with FITC-conjugated
anti-CD14 MAb. Cells were suspended in HEPES-buffered isotonic KCI medium with no added Ca2⫹. Ethidium (25
␮M) was added 1 min before 1.0 mM ATP (arrow). Cells were gated on monocytes population by forward and side
scatter, and only those positive for CD14 cells were selected for analysis. Ethidium uptake was measured at 5-s
intervals. Shaded area represents the range for normal monocytes. D: 3⬘-O-(4 benzoylbenzoyl)-ATP (BzATP)-induced
ethidium uptake into B-lymphocytes from patients CLL 2 (functional P2X7) and CLL 4 (nonfunctional P2X7) in
Na⫹-free, low-Cl⫺ medium. Cells were first stained with FITC-conjugated anti-CD19 MAb and then washed and
resuspended in isotonic sucrose medium (280 mM sucrose, 5 mM KCI, and 20 mM HEPES, pH 7.4) at 37°C. Ethidium
was added, followed 40 s later by 100 ␮M BzATP (arrow). Mean channel of cell-associated fluorescence intensity was
measured for gated CD19⫹ lymphocyte populations at 5-s intervals.
etic origin that express this receptor in its native state.
Both these studies and those in HEK-293 cells heterologously expressing the cDNA for P2X7 have revealed
features that are most unusual for a channel. These
include the slow kinetics of channel dilatation and the
triggering of downstream events such as activation
of membrane metalloprotease(s) and intracellular
caspase activation leading to apoptosis. Recently, a
FUNCTIONAL AND NONFUNCTIONAL P2X7 RECEPTORS IN LEUCOCYTES
Fig. 5. Agarose gel electrophoresis (2% agarose) of RT-PCR products
using human P2X7-specific primers. cDNA was from fluorescenceactivated cell sorter (FACS)-purified B-CLL lymphocytes prepared
from 6 different patients. A single 495-bp product is shown at the
predicted position for P2X7.
monoclonal antibody against an extracellular epitope
of the P2X7 receptor was developed (2) that was used in
this study for flow cytometric analysis of receptor expression on the various cells in peripheral blood. P2X7
receptor expression was approximately four- to fivefold
greater on monocytes than on normal or leukemic B
lymphocytes, whereas there were no significant differences among the three main lymphocyte subsets of T,
B, and NK cells. Little or no surface expression of P2X7
receptor was found on either polymorphonuclear neutrophils or blood platelets. The same rank order of cell
surface P2X7 receptor function was found by measuring initial rates over 5 min of ATP-induced uptake of
ethidium into these various cell types within the one
mononuclear cell population. This functional assay
made use of two-color flow cytometry in which monocytes and NK, B, and T lymphocytes were identified by
FITC- or RPE-Cy5-labeled antibodies against CD14,
CD16, CD19, and CD3, respectively, whereas ethidium
uptake was measured on the red (570 nm) channel. A
close correlation was found between expression of P2X7
receptors and the ATP-induced ethidium uptake into
the various leucocyte types, suggesting that all P2X7
receptors expressed on the surface of normal lymphocytes and monocytes are functionally competent. Previous studies have suggested that P2X7 is not expressed in freshly isolated monocytes and that receptor
expression requires differentiation of these cells to
macrophages under the influence of interferon-␥ or
lipopolysaccharide. (2, 24). However, our data for both
expression (Fig. 1) and function (Fig. 2) clearly show
functional P2X7 receptors on the surface of all fresh
circulating blood monocytes. The large size and surface
area of the monocyte (ca. 500 fl) may explain in part
their four- to fivefold greater number of P2X7 receptors
than are found on the smaller lymphocytes (180–210
fl). The greater surface area of monocytes is also shown
by their expression of threefold more membrane sodium pumps than lymphocytes as measured by the
binding of the cardiac glycoside [3H]ouabain (49). Nevertheless, monocytes have the capacity to greatly upregulate their P2X7 function on differentiation to macrophages under the influence of interferon-␥ (1, 24).
The high expression of intracellular P2X7 protein is
of interest and appears to be a ubiquitous finding in
cells of hemopoietic origin, although erythrocytes were
C1195
not tested for technical reasons. The finding that most
of the P2X7 receptor is in an intracellular location may
have parallels to other receptors such as those for
insulin and for the chemokine receptor CXCR4. Both
these receptors undergo endosomal internalization following receptor occupancy by their respective agonists
(9, 26, 38). Recently, it has been demonstrated that the
P2Y2 nucleotide receptor can also undergo internalization following binding of its ligand, UTP (20, 39), although it is uncertain whether this receptor then undergoes recycling back to the cell surface as shown for
the insulin receptor of adipose cells. Likewise, a rapid
internalization of the P2X1 receptor has been demonstrated by confocal microscopy on exposure of smooth
muscle cells to ATP (11). The intracellular pool of P2X7
receptors such as on neutrophils or platelets (Fig. 1) is
atypical because there is little cell surface expression
on these cell types. Perhaps this intracellular receptor
pool forms a reserve that is able to be recruited to the
surface following cellular activation.
A main finding is that lymphocytes from three BCLL subjects showed strong P2X7 immunoreactivity on
the lymphocyte surface (Fig. 3) but no ATP-induced
ethidium uptake and very poor ATP-induced Ba2⫹ uptake (Fig. 4). There is little information on the factors
that modulate the surface expression and/or function of
the P2X7 channel. Sodium media are known to partially inhibit the function of P2X7 (47). However, the
assays shown in Fig. 4, A and B, were performed in K⫹rather than Na⫹-based media to avoid any inhibitory
effect of Na⫹. Chloride ions also partially inhibit the
function of P2X7 (30), but even a Na⫹-free, low-Cl⫺
medium with BzATP as agonist did not correct the
poorly functional P2X7. Hormones may also influence
the expression and/or function of P2X7, because the
cytokine interferon-␥ has been shown to upregulate
P2X7 receptor function during maturation of monocyte
to macrophages (1, 24). A recent study of B-CLL has
Fig. 6. Time course of BzATP-induced loss of L-selectin. B-CLL
lymphocytes with functionally active (F) and inactive (E) P2X7 receptor were incubated with 100 ␮M BzATP for the indicated times
before being labeled with anti-L-selectin antibody. Results are
means ⫾ SE and are expressed as the percentage of initial expression (n ⫽ 5 for functional P2X7 and n ⫽ 3 for nonfunctional P2X7
patients).
C1196
FUNCTIONAL AND NONFUNCTIONAL P2X7 RECEPTORS IN LEUCOCYTES
shown elevated values for tumor necrosis factor
(TNF)-␣ and IL-8 in the serum of these patients (16,
17). However, the effect of neither TNF-␣ nor IL-8 on
P2X7 expression on lymphocytes is known. Incubation
up to 48 h of lymphocytes from patients with nonfunctional P2X7 in buffered medium containing 80%
plasma from patients with functional P2X7 failed to
produce a P2X7 response. Also, P2X7 functional responses were not inhibited by prolonged incubation of
responding B-CLL cells in plasma from patients with
nonfunctional P2X7 (unpublished observations). Buell
and colleagues (3) showed that ambient ATP released
during incubation of HL-60 cells produces a sustained
desensitization of P2X1 receptors but that apyrase
added to the medium can resensitize this receptor.
However, addition of apyrase (5 U/ml) during incubation of B-CLL lymphocytes failed to reactivate the
functionally inactive P2X7 in these cells (unpublished
observations). Although we have been unable to find
evidence for a soluble inhibitory factor responsible for
the decrease in P2X7 function, it is intriguing that
monocytes from these same patients also showed an
impaired P2X7 function (Fig. 4C). This finding strongly
suggests that cell-cell contact or some short-range mediator impairs P2X7 function following the many cellular interactions that occur during the traffic of these
cells through the lymph node. An alternate possibility
is that genetic changes may underlie the loss of function of the P2X7 receptor. Both point mutations and
nonrandom chromosomal changes are associated with
B-CLL involving trisomy of chromosome 12 in 20% of
cases (8, 40). Genomic instability of chromosome 12 is
common in B-CLL (27, 29) and involves the regions
(12q 21–23) adjoining the location of P2X7 on 12q 24
(4). Although our RT-PCR analyses (Fig. 5) exclude a
major genetic deletion causing truncation of the long
COOH-terminal tail of the P2X7 receptor, it is still
possible that point mutations or deletions in other
parts of the receptor may underlie the impaired P2X7
function in certain patients.
Lymphocytes in the body undergo continuous recirculation between the blood and tissues (21), and the
first step of lymphocyte emigration to the lymph node
is its adhesion to vascular endothelial cells. The initial
event in this adhesion involves the interaction of Lselectin with counterreceptors on the surface of endothelial cells followed by transendothelial migration.
The central importance of L-selectin in regulating leucocyte emigration has been shown in L-selectin “knockout mice,” which show greatly reduced lymphocyte
numbers in lymph nodes and an impaired ability of
leucocytes to emigrate to sites of inflammation (44). It
is known that extracellular ATP acting via the lymphocyte P2X7 receptor can activate a membrane metalloproteinase that cleaves L-selectin at a membrane-proximal site to release soluble L-selectin (23, 25).
However, the lymphocytes from those B-CLL subjects
who have functionally inactive P2X7 receptors failed to
show L-selectin downregulation with extracellular
ATP (Fig. 6). These subjects have high levels of Lselectin expression on their lymphocytes, which con-
trasts with the low levels of L-selectin that are generally found on the surface of B-CLL lymphocytes with
functional P2X7 (6). This study has shown that the
functional state of P2X7 receptors regulates the expression of the homing adhesion molecule L-selectin,
whereas in contrast, the B cell chemoattractant SDF
(stromal cell-derived factor)-1␣ has little effect on this
adhesion molecule (Gu BJ, Dao LP, and Wiley JS,
unpublished observations). It is likely that the functional status of P2X7 receptors plays a central role in
regulating L-selectin expression on lymphocytes and in
directing their patterns of recirculation in the body.
We are grateful to Drs. Julian Barden, Arthur Conigrave, and
Michael Morris for helpful discussions, Mary Sartor for FACS cell
sorting, Phuong Dao for immunophenotyping, and Shelley Spicer for
typing the manuscript.
This work was supported by the National Health and Medical
Research Council of Australia, the NSW Cancer Council, the Leo &
Jenny Foundation, and a Sydney University Cancer Research Grant.
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