1 Department of Medicine, Nepean Hospital, University of Sydney, Penrith, New South Wales 2750, Australia; 2 Department of Pharmacology, University of Cambridge, Cambridge CB2 1QJ, United Kingdom; and 3 Department of Molecular Biology, Geneva Institute for Biomedical Research, 1228 Geneva, Switzerland
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ABSTRACT |
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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
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INTRODUCTION |
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RECEPTORS 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 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. 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.
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MATERIALS AND METHODS |
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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). Ficoll-Hypaque (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 FITC-labeled 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 µl of 1.0 × 107 cells/ml in HEPES-buffered NaCl medium) or PRP (100 µl of 1-2 × 108 cells/ml) were incubated with fluorescence-conjugated anti-P2X7 antibody (60 µg/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 µM) 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).
Cytosolic Ba2+ measurements by fluorometry. Lymphocytes (1 × 107 cells/ml) were washed once and loaded with 2 µM 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 µM 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 µg of total RNA was used in the RT reaction with 0.5 µg 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 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 µl of RNase A was added, and the tube was incubated for 20 min at 37°C. To amplify the COOH-terminal 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-µl reaction contained 1× PCR buffer, 1.5 mM MgCl2, 100 µM 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 |
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Surface expression of P2X7 on normal lymphocytes and
monocytes.
In Fig. 1, the surface expression of
P2X7 was measured by flow cytometry of normal white cells
stained with directly conjugated P2X7 antibodies.
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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).
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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 B-CLL lymphocytes (Table 1, P < 0.001).
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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 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.
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Correlation of surface P2X7 expression and ATP-induced 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
COOH-terminal 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).
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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.
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DISCUSSION |
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Much of the characterization of the P2X7 receptor
(previously termed P2Z) has been in cells of hemopoietic 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 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 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 B-CLL 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 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
L-selectin 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 L-selectin expression on their
lymphocytes, which contrasts 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
Address for reprint requests and other correspondence: J. S. Wiley, Clinical Sciences Bldg., Nepean Hospital, Penrith, NSW 2750, Australia (E-mail: wileyj{at}medicine.usyd.edu.au).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 8 December 1999; accepted in final form 26 April 2000.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Blanchard, DK,
McMillen S,
and
Djeu JY.
IFN-gamma enhances sensitivity of human macrophages to extracellular ATP-mediated lysis.
J Immunol
147:
2579-2585,
1991
2.
Buell, G,
Chessell IP,
Michel AD,
Collo G,
Salazzo M,
Herren S,
Gretener D,
Grahames C,
Kaur R,
Kosco-Vilbois MH,
and
Humphrey PP.
Blockade of human P2X7 receptor function with a monoclonal antibody.
Blood
92:
3521-3528,
1998
3.
Buell, G,
Michel AD,
Lewis C,
Collo G,
Humphrey PP,
and
Surprenant A.
P2X1 receptor activation in HL60 cells.
Blood
87:
2659-2664,
1996
4.
Buell, GN,
Talabot F,
Gos A,
Lorenz J,
Lai E,
Morris MA,
and
Antonarakis SE.
Gene structure and chromosomal localization of the human P2X7 receptor.
Receptors Channels
5:
347-354,
1998[ISI][Medline].
5.
Burnstock, G.
P2 purinoceptors: historical perspective and classification.
Ciba Found Symp
198:
1-28,
1996[ISI][Medline].
6.
Chen, JR,
Gu BJ,
Dao LP,
Bradley CJ,
Mulligan SP,
and
Wiley JS.
Transendothelial migration of lymphocytes in chronic lymphocytic leukaemia is impaired and involved down-regulation of both L-selectin and CD23.
Br J Haematol
105:
181-189,
1999[ISI][Medline].
7.
Coutinho-Silva, R,
Persechini PM,
Bisaggio RD,
Perfettini JL,
Neto AC,
Kanellopoulos JM,
Motta-Ly I,
Dautry-Varsat A,
and
Ojcius DM.
P2Z/P2X7 receptor-dependent apoptosis of dendritic cells.
Am J Physiol Cell Physiol
276:
C1139-C1147,
1999
8.
Criel, A,
Verhoef G,
Vlietinck R,
Mecucci C,
Billiet J,
Michaux L,
Meeus P,
Louwagie A,
Van Orshoven A,
Van Hoof A,
Boogaerts M,
Van den Berghe H,
and
De Wolf-Peeters C.
Further characterization of morphologically defined typical and atypical CLL: a clinical, immunophenotypic, cytogenetic and prognostic study on 390 cases.
Br J Haematol
97:
383-391,
1997[ISI][Medline].
9.
Di Guglielmo, GM,
Drake PG,
Baass PC,
Authier F,
Posner BI,
and
Bergeron JJ.
Insulin receptor internalization and signalling.
Mol Cell Biochem
182:
59-63,
1998[ISI][Medline].
10.
Dubyak, GR,
and
el-Moatassim C.
Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides.
Am J Physiol Cell Physiol
265:
C577-C606,
1993
11.
Dutton JL, Poronnik P, Li GH, Holding C, Worthington RA, Vandenberg RI,
Cook DI, Barden JA, and Bennett MA. P2X1 receptor
membrane distribution and down-regulation visualized using receptor
coupled green fluorescent protein. Neuropharmacology. In
press.
12.
El-Moatassim, C,
and
Dubyak GR.
A novel pathway for the activation of phospholipase D by P2z purinergic receptors in BAC1.2F5 macrophages.
J Biol Chem
267:
23664-23673,
1992
13.
Fernando, KC,
Gargett CE,
and
Wiley JS.
Activation of the P2Z/P2X7 receptor in human lymphocytes produces a delayed permeability lesion: involvement of phospholipase D.
Arch Biochem Biophys
362:
197-202,
1999[ISI][Medline].
14.
Ferrari, D,
Los M,
Bauer MK,
Vandenabeele P,
Wesselborg S,
and
Schulze-Osthoff K.
P2Z purinoreceptor ligation induces activation of caspases with distinct roles in apoptotic and necrotic alterations of cell death.
FEBS Lett
447:
71-75,
1999[ISI][Medline].
15.
Ferrari, D,
Wesselborg S,
Bauer MA,
and
Schulze-Osthoff K.
Extracellular ATP activates transcription factor NF-kappaB through the P2Z purinoreceptor by selectively targeting NF-kappaB p65.
J Cell Biol
139:
1635-1643,
1997
16.
Foa, R,
Massaia M,
Cardona S,
Tos AG,
Bianchi A,
Attisano C,
Guarini A,
di Celle PF,
and
Fierro MT.
Production of tumor necrosis factor-alpha by B-cell chronic lymphocytic leukemia cells: a possible regulatory role of TNF in the progression of the disease.
Blood
76:
393-400,
1990[Abstract].
17.
Francia di Celle, P,
Mariani S,
Riera L,
Stacchini A,
Reato G,
and
Foa R.
Interleukin-8 induces the accumulation of B-cell chronic lymphocytic leukemia cells by prolonging survival in an autocrine fashion.
Blood
87:
4382-4389,
1996
18.
Gargett, CE,
Cornish EJ,
and
Wiley JS.
Phospholipase D activation by P2Z-purinoceptor agonists in human lymphocytes is dependent on bivalent cation influx.
Biochem J
313:
529-535,
1996[ISI][Medline].
19.
Gargett, CE,
Cornish JE,
and
Wiley JS.
ATP, a partial agonist for the P2Z receptor of human lymphocytes.
Br J Pharmacol
122:
911-917,
1997[Abstract].
20.
Garrad, RC,
Otero MA,
Erb L,
Theiss PM,
Clarke LL,
Gonzalez FA,
Turner JT,
and
Weisman GA.
Structural basis of agonist-induced desensitization and sequestration of the P2Y2 nucleotide receptor. Consequences of truncation of the C terminus.
J Biol Chem
273:
29437-29444,
1998
21.
Gowans, JL.
The recirculation of lymphocytes from blood to lymph in the rat.
J Physiol (Lond)
146:
54-69,
1959[ISI].
22.
Greenberg, S,
Di Virgilio F,
Steinberg TH,
and
Silverstein SC.
Extracellular nucleotides mediate Ca2+ fluxes in J774 macrophages by two distinct mechanisms.
J Biol Chem
263:
10337-10343,
1988
23.
Gu, B,
Bendall LJ,
and
Wiley JS.
Adenosine triphosphate-induced shedding of CD23 and L-selectin (CD62L) from lymphocytes is mediated by the same receptor but different metalloproteases.
Blood
92:
946-951,
1998
24.
Hickman, SE,
el Khoury J,
Greenberg S,
Schieren I,
and
Silverstein SC.
P2Z adenosine triphosphate receptor activity in cultured human monocyte-derived macrophages.
Blood
84:
2452-2456,
1994
25.
Jamieson, GP,
Snook MB,
Thurlow PJ,
and
Wiley JS.
Extracellular ATP causes of loss of L-selectin from human lymphocytes via occupancy of P2Z purinoceptors.
J Cell Physiol
166:
637-642,
1996[ISI][Medline].
26.
Kandror, KV.
Insulin regulation of protein traffic in rat adipose cells.
J Biol Chem
274:
25210-25217,
1999
27.
Karhu, R,
Knuutila S,
Kallioniemi OP,
Siltonen S,
Aine R,
Vilpo L,
and
Vilpo J.
Frequent loss of the 11q14-24 region in chronic lymphocytic leukemia: a study by comparative genomic hybridization. Tampere CLL Group.
Genes Chromosomes Cancer
19:
286-290,
1997[ISI][Medline].
28.
Markwardt, F,
Lohn M,
Bohm T,
and
Klapperstuck M.
Purinoceptor-operated cationic channels in human B lymphocytes.
J Physiol (Lond)
498:
143-151,
1997[Abstract].
29.
Merup, M,
Juliusson G,
Wu X,
Jansson M,
Stellan B,
Rasool O,
Roijer E,
Stenman G,
Gahrton G,
and
Einhorn S.
Amplification of multiple regions of chromosome 12, including 12q13-15, in chronic lymphocytic leukaemia.
Eur J Haematol
58:
174-180,
1997[ISI][Medline].
30.
Michel, AD,
Chessell IP,
and
Humphrey PP.
Ionic effects on human recombinant P2X7 receptor function.
Naunyn Schmiedebergs Arch Pharmacol
359:
102-109,
1999[ISI][Medline].
31.
Naumov, AP,
Kaznacheyeva EV,
Kiselyov KI,
Kuryshev YA,
Mamin AG,
and
Mozhayeva GN.
ATP-activated inward current and calcium-permeable channels in rat macrophage plasma membranes.
J Physiol (Lond)
486:
323-337,
1995[Abstract].
32.
Nicke, A,
Baumert HG,
Rettinger J,
Eichele A,
Lambrecht G,
Mutschler E,
and
Schmalzing G.
P2X1 and P2X3 receptors form stable trimers: a novel structural motif of ligand-gated ion channels.
EMBO J
17:
3016-3028,
1998
33.
Nuttle, LC,
and
Dubyak GR.
Differential activation of cation channels and non-selective pores by macrophage P2z purinergic receptors expressed in Xenopus oocytes.
J Biol Chem
269:
13988-13996,
1994
34.
Perregaux, DG,
and
Gabel CA.
Post-translational processing of murine IL-1: evidence that ATP-induced release of IL-1 alpha and IL-1 beta occurs via a similar mechanism.
J Immunol
160:
2469-2477,
1998
35.
Pizzo, P,
Zanovello P,
Bronte V,
and
Di Virgilio F.
Extracellular ATP causes lysis of mouse thymocytes and activates a plasma membrane ion channel.
Biochem J
274:
139-144,
1991[ISI][Medline].
36.
Saribas, AS,
Lustig KD,
Zhang X,
and
Weisman GA.
Extracellular ATP reversibly increases the plasma membrane permeability of transformed mouse fibroblasts to large macromolecules.
Anal Biochem
209:
45-52,
1993[ISI][Medline].
37.
Schilling, WP,
Rajan L,
and
Strobl-Jager E.
Characterization of the bradykinin-stimulated calcium influx pathway of cultured vascular endothelial cells. Saturability, selectivity, and kinetics.
J Biol Chem
264:
12838-12848,
1989
38.
Signoret, N,
Oldridge J,
Pelchen-Matthews A,
Klasse PJ,
Tran T,
Brass LF,
Rosenkilde MM,
Schwartz TW,
Holmes W,
Dallas W,
Luther MA,
Wells TN,
Hoxie JA,
and
Marsh M.
Phorbol esters and SDF-1 induce rapid endocytosis and down modulation of the chemokine receptor CXCR4.
J Cell Biol
139:
651-664,
1997
39.
Sromek, SM,
and
Harden TK.
Agonist-induced internalization of the P2Y2 receptor.
Mol Pharmacol
54:
485-494,
1998
40.
Su'ut, L,
O'Connor SJ,
Richards SJ,
Jones RA,
Roberts BE,
Davies FE,
Fegan CD,
Jack AS,
and
Morgan GJ.
Trisomy 12 is seen within a specific subtype of B-cell chronic lymphoproliferative disease affecting the peripheral blood/bone marrow and co-segregates with elevated expression of CD11a.
Br J Haematol
101:
165-170,
1998[ISI][Medline].
41.
Surprenant, A,
Buell G,
and
North RA.
P2X receptors bring new structure to ligand-gated ion channels.
Trends Neurosci
18:
224-229,
1995[ISI][Medline].
42.
Surprenant, A,
Rassendren F,
Kawashima E,
North RA,
and
Buell G.
The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7).
Science
272:
735-738,
1996[Abstract].
43.
Tatham, PE,
and
Lindau M.
ATP-induced pore formation in the plasma membrane of rat peritoneal mast cells.
J Gen Physiol
95:
459-476,
1990[Abstract].
44.
Tedder, TF,
Steeber DA,
and
Pizcueta P.
L-selectin-deficient mice have impaired leukocyte recruitment into inflammatory sites.
J Exp Med
181:
2259-2264,
1995[Abstract].
45.
Wiley, JS,
Chen JR,
Snook MB,
and
Jamieson GP.
The P2Z-purinoceptor of human lymphocytes: actions of nucleotide agonists and irreversible inhibition by oxidized ATP.
Br J Pharmacol
112:
946-950,
1994[Abstract].
46.
Wiley, JS,
Chen R,
and
Jamieson GP.
The ATP4 receptor-operated channel (P2Z class) of human lymphocytes allows Ba2+ and ethidium+ uptake: inhibition of fluxes by suramin.
Arch Biochem Biophys
305:
54-60,
1993[ISI][Medline].
47.
Wiley, JS,
Chen R,
Wiley MJ,
and
Jamieson GP.
The ATP4 receptor-operated ion channel of human lymphocytes: inhibition of ion fluxes by amiloride analogs and by extracellular sodium ions.
Arch Biochem Biophys
292:
411-418,
1992[ISI][Medline].
48.
Wiley, JS,
Gargett CE,
Zhang W,
Snook MB,
and
Jamieson GP.
Partial agonists and antagonists reveal a second permeability state of human lymphocyte P2Z/P2X7 channel.
Am J Physiol Cell Physiol
275:
C1224-C1231,
1998
49.
Wiley, JS,
Kraft N,
and
Cooper IA.
The binding of ouabain to normal and chronic lymphocytic leukemic lymphocytes.
Blood
54:
994-1000,
1979[Abstract].
50.
Yamaguchi, DT,
Green J,
Kleeman CR,
and
Muallem S.
Properties of the depolarization-activated calcium and barium entry in osteoblast-like cells.
J Biol Chem
264:
197-204,
1989
51.
Zheng, LM,
Zychlinsky A,
Liu CC,
Ojcius DM,
and
Young JD.
Extracellular ATP as a trigger for apoptosis or programmed cell death.
J Cell Biol
112:
279-288,
1991[Abstract].