Human leukocytes express a receptor for extracellular nucleotides, named P2X7R, that in lymphocytes can either mediate cell death or proliferation, depending on the level of activation. The authors have investigated P2X7R expression and function in 21 patients affected by B-cell chronic lymphocytic leukemia, 13 with an evolutive and 8 with an indolent variant of the disease. Resting cytoplasmic Ca++ concentration was significantly higher in lymphocytes from patients with the evolutive compared with indolent variant. Furthermore, in the former, P2X7R stimulation triggered a Ca++ influx significantly larger. Higher Ca++ influx correlated with an increased P2X7R expression in the lymphocytes from patients with the evolutive form. Finally, incubation in the presence of extracellular adenosine triphosphate decreased spontaneous proliferation of lymphocytes from patients affected with the evolutive variant but had no effects on lymphocytes from patients with the indolent form. These results suggest that expression and function of P2X7R may correlate with the severity of B-cell chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (B-CLL) is one of the most common hemopoietic tumors.1,2 Patients with B-CLL experience heterogenous clinical courses: some survive for more than a decade with minimal therapy, while others die within a few years despite aggressive treatment. Therefore, the identification of novel markers that may serve as prognostic indicators might be of great help. B-CLL cells, in striking contrast with normal B lymphocytes, have a high level of expression of the nucleotide P2X7 receptor (P2X7R).3-5 This ligand (adenosine triphosphate [ATP])–gated plasma membrane channel is present in immune cells,6 neurons, fibroblasts, epithelia, and smooth muscle cells.7 Its physiologic function is basically unknown. However, there is little doubt that protracted stimulation causes cell death either by apoptosis or necrosis, depending on the cell type and the experimental conditions.8-10 Peripheral T lymphocytes and monocytes express P2X7R11,12even if in some patients this receptor may be nonfunctional.13 

We have recently observed that transfection of P2X7R into human P2X7null lymphoblastoid cells renders these cells capable of growth in serum-depleted culture medium and that proliferation relies on the continuous release of ATP (4-fold larger in the P2X7 transfectants compared with mock-transfected cells).14 In the present report we show that P2X7R is differentially expressed in patients with B-CLL with evolutive B-CLL courses versus indolent B-CLL courses and that in vitro proliferation of lymphocytes isolated from evolutive B-CLL patients is inhibited by extracellular ATP.

Patients

Twenty-one patients with typical CLL, according to morphologic and immunologic criteria,15 seen at the Section of Hematology, University of Ferrara, during the study period were included in this analysis. All patients were CD5+/CD23+, with weak expression of CD22 and surface immunoglobulins. The patients did not receive cytotoxic therapy for at least 2 months before sampling. All patients had a follow-up of at least 3 years with hematologic workups at 1- to 6-month intervals: the patients here referred to as affected by an “indolent” disease were those who never required cytotoxic therapy, with stable blood counts and no disease progression, whereas those patients with therapy-demanding disease due to a shorter than 12-month lymphocyte doubling time, or to disease progression through the Rai staging system, were classified as “evolutive.” The karyotype was abnormal in 3 of 8 patients (12p+, 14q+ −21 in 1 patient each) in the former group and in 6 of 13 patients in the latter group (11q22-23 abnormality in 3 patients and 13q, 18p+, and t(1;6)(q25;p24) in 1 patient each). Approval was obtained from the Institutional Review Board for these studies. Informed consent was provided according to the Declaration of Helsinki.

Cells

Peripheral lymphocytes were isolated by Ficoll gradient and resuspended in RPMI medium at a concentration of 107/mL prior to experiments.

Measurement of the cytoplasmic free Ca++concentration

Measurements were performed in a thermostat-controlled and magnetically stirred Perkin-Elmer fluorimeter (Beaconsfield, United Kingdom) with the fluorescent indicator Fura-2/AM as previously described14 in a solution containing 300 mM sucrose, 1 mM MgCl2, 1 mM K2HPO4, 5 mM KHCO3, 5.5 mM glucose, 1 mM CaCl2, and 20 mM HEPES (pH adjusted to 7.4 with KOH).

Reverse-transcriptase–polymerase chain reaction

Total cytoplasmic RNA was extracted as described by Chomczynski and Sacchi,16 reverse transcribed, and amplified with P2X7-specific primers.14 After hybridization with a digoxigenin-labeled P2X7-specific internal oligoprobe, P2X7 complementary DNA was visualized by chemiluminescent detection after incubation with a dilution of antidigoxigenin Fab fragments conjugated to alkaline phosphatase. As a control for the mRNA content of the samples, β-actin was used. The β-actin amplification products are shown as ethidium bromide–stained agarose gel electrophoresis bands.

Western blotting

Cells were lysed in lysis buffer containing 300 mM sucrose, 1 mM K2HPO4, 1 mM MgSO4, 5.5 mM glucose, 20 mM HEPES (pH 7.4), 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 0.2 μg deoxyribonuclease, and 0.2 μg ribonuclease by repeated freeze/thawing (3 cycles). Proteins were separated on 7.5% sodium dodecyl sulfate–polyacrylamide gel. The rabbit polyclonal anti-P2X7 receptor serum was kindly provided by Dr Gary Buell (Serono Pharmaceutical Research Institute, Geneva, Switzerland). The primary antibody was used at a dilution of 1:100 in triethanolamine-buffered saline buffer (10 mM Tris-HCl, 150 mM NaCl, pH 8.0). The secondary antibody was a goat antirabbit antibody conjugated to alkaline phosphatase. Intensity of the bands was measured with a Multimage Light Cabinet Alpha Image 1220 densitometer (Alpha Innotech, San Leandro, CA) and expressed as integrated density value.

Proliferation

For [3H]thymidine incorporation, cells were resuspended in RPMI supplemented with 10% fetal calf serum and plated overnight at a concentration of 106/mL in the presence of radiolabeled thymidine (1 μCi [3.7 × 104 Bq] per well). In parallel, cells were also counted with a phase contrast light microscope.

Statistics

Statistical significance was assessed by the Studentt test.

It was previously reported by Wiley and Dubyak3 that ATP increases cation permeability of lymphocytes from patients affected by B-CLL but not from healthy patients. We have reevaluated their findings in 2 groups of patients—1 with an evolutive and 1 with an indolent course of the disease. Exemplificative traces of resting and ATP-stimulated levels of intracellular Ca++([Ca++]i) are shown in Figure1. Lymphocytes from evolutive B-CLL showed an elevated resting [Ca++]i and a higher ATP-stimulated peak compared with the patient with the indolent variant. We screened 13 evolutive B-CLLs and 8 indolent B-CLLs, finding a mean resting [Ca++]i of 88 nm ± 27 and 52 nm ± 23 (P < .01), respectively. The stimulated [Ca++]i increase above resting level was 100 nm ± 30 and 32 nm ± 16 (P < .001) in evolutive B-CLL and indolent B-CLL, respectively. There was a good correlation between an elevated basal [Ca++]i and a higher ATP-stimulated rise (r = 0.63). We confirmed, as previously shown by Wiley and Dubyak,3 that B-CLL cells do not express functional P2Y receptors and that the whole [Ca++]i increase is due to influx across the plasma membrane following the opening of an ATP-gated ion channel (Figure 1). Because previous studies showed that the main ATP-activated channel of lymphoblastoid cells is P2X7R,5,17,18 we investigated the expression of this receptor by reverse-transcriptase–polymerase chain reaction (RT-PCR) in peripheral lymphocytes from all the patients recruited in the 2 groups. Figure 2A reports P2X7R expression in 2 evolutive B-CLL and 2 indolent B-CLL patients representative of the respective groups; human macrophages are shown for comparison. Control β-actin amplification fragments are shown in Figure 2B. Lymphocytes from patients with evolutive B-CLL express P2X7R to a level higher than the patients affected by the indolent variant, and comparable to that of macrophages, a cell type well known for its high expression of this receptor subtype. Patients with a higher ATP-stimulated [Ca++]ishowed a higher P2X7R expression (Figure 2A). Figure 2C shows a Western blot of peripheral lymphocytes from the same patients to confirm higher expression of the P2X7R protein in evolutive compared with indolent B-CLL. In our screening of all the 21 patients recruited in this study, we found a good positive correlation (r = 0.924, P < .01) between increases in [Ca++]i stimulated by ATP and P2X7R protein expression measured by densitometry of Western blots. We and others have demonstrated in the past that expression of P2X7R confers susceptibility to ATP-mediated cytotoxicity.10 19 Thus, we selected 8 patients—4 B-CLL indolent (low P2X7R) and 4 B-CLL evolutive (high P2X7R)—and tested their sensitivity to ATP. In the experiment reported in Figure 2D, we show that extracellular ATP inhibited in vitro proliferation of peripheral lymphocytes isolated from patients affected by the evolutive but not the indolent form of the disease. Data on [3H]thymidine incorporation were corroborated by cell number count (not shown).

Fig. 1.

ATP-triggered lymphocyte [Ca++] increase.

ATP triggers a higher [Ca++]iincrease in lymphocytes from evolutive (A) compared with indolent (B) B-CLL. Lymphocytes were isolated as described in “Study design” and resuspended in Ca++-supplemented or Ca++-free solution at a concentration of 2 × 106/mL. The Ca++-free solution also contained 500 μM EGTA. ATP and ionomycin (iono) were 1 mM and 1 μM, respectively.

Fig. 1.

ATP-triggered lymphocyte [Ca++] increase.

ATP triggers a higher [Ca++]iincrease in lymphocytes from evolutive (A) compared with indolent (B) B-CLL. Lymphocytes were isolated as described in “Study design” and resuspended in Ca++-supplemented or Ca++-free solution at a concentration of 2 × 106/mL. The Ca++-free solution also contained 500 μM EGTA. ATP and ionomycin (iono) were 1 mM and 1 μM, respectively.

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Fig. 2.

Lymphocytes from evolutive B-CLL express a high level of P2X7R and are inhibited by ATP.

(A) RT-PCR amplification and detection were performed as described in “Study design”; 10 μL PCR product was loaded in each lane. Amplification products from 2 evolutive (E1 and E2) or indolent (I1 and I2) B-CLL patients are shown; human macrophages are shown as a control (Cont). In these same lymphocyte populations, increases in [Ca++]i ([Ca++]i) caused by stimulation with 1 mM ATP were also measured and reported below each lane. [Ca++]i increases are expressed as means ± SD of triplicate determinations. (B) β-actin amplification fragments. (C) Western blot with a specific anti-P2X7 serum (see “Study design”). (D) Lymphocytes, 106 per well, from 8 patients (4 B-CLL indolent and 4 B-CLL evolutive) were incubated as described in “Study design” for 24 hours in the presence or absence of 2.5 mM ATP. Data (cpm) are the means ± SD of quadruplicate determinations from a single experiment representative of 4 others. Statistical significance (P) between evolutive B-CLL incubated without or with ATP is reported above bars.

Fig. 2.

Lymphocytes from evolutive B-CLL express a high level of P2X7R and are inhibited by ATP.

(A) RT-PCR amplification and detection were performed as described in “Study design”; 10 μL PCR product was loaded in each lane. Amplification products from 2 evolutive (E1 and E2) or indolent (I1 and I2) B-CLL patients are shown; human macrophages are shown as a control (Cont). In these same lymphocyte populations, increases in [Ca++]i ([Ca++]i) caused by stimulation with 1 mM ATP were also measured and reported below each lane. [Ca++]i increases are expressed as means ± SD of triplicate determinations. (B) β-actin amplification fragments. (C) Western blot with a specific anti-P2X7 serum (see “Study design”). (D) Lymphocytes, 106 per well, from 8 patients (4 B-CLL indolent and 4 B-CLL evolutive) were incubated as described in “Study design” for 24 hours in the presence or absence of 2.5 mM ATP. Data (cpm) are the means ± SD of quadruplicate determinations from a single experiment representative of 4 others. Statistical significance (P) between evolutive B-CLL incubated without or with ATP is reported above bars.

Close modal

Receptors for extracellular nucleotides have recently become a focus of interest in immunology and hematology due to their high level of expression in blood cells and the vast potential of therapeutic applications afforded by their modulation.6 It was originally put forward that P2X7R might participate in shedding of CD23 and CD62L or killing by apoptosis.4,20,21We have previously observed that transfection of P2X7R, the most widely diffused P2X receptor subtype in immune cells, confers a substantial proliferative advantage when transfected into human B lymphoblastoid cells that lack it constitutively.14 The P2X7R transfectants become able to grow in the absence of serum, show an elevated resting [Ca++]i, respond to ATP, and secrete large amounts of this nucleotide into the culture supernatant. Thus, we speculated that P2X7R expression might also confer an advantage in vivo and anticipated that patients with the worse prognosis (ie, evolutive B-CLL) might be characterized by a B-cell population with a higher P2X7R expression. Such a cell population might be less affected than normal B lymphocytes, or B-CLLs that express low levels of P2X7R (ie, indolent B-CLL cells), by antimetabolites or might recover more quickly after chemotherapy. It is of interest that while P2X7R expression confers to evolutive B-CLL cells a proliferation advantage, it also makes these cells more susceptible to the cytotoxic effect of high concentrations of extracellular ATP. It may appear paradoxical that stimulation of a single receptor may bring about such utterly different effects, but this might be explained by the different pattern of intracellular signals generated by a tonic, low-level activation of the P2X7R, such as that presumably responsible for growth stimulation, as opposed to the massive receptor activation that is known to trigger cytotoxicity. In any case, this bifunctional capacity points to P2X7R as a novel and useful target for antitumor therapy.

Supported by grants by the Italian Ministry of Scientific Research (ex 40%), the University of Ferrara, the National Research Council of Italy (Target Project on Biotechnology), the Italian Association for Cancer Research, the Istituto Superiore di Sanità (ISS Grant on Tumor Therapy), the Italian Space Agency (ASI), and Telethon of Italy.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

1
Landis
SH
Murray
T
Bolden
S
Wingo
PA
Cancer statistics.
CA Cancer J Clin.
48
1998
6
29
2
Damle
RN
Wasil
T
Fais
F
et al
Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia.
Blood.
94
1999
1840
1847
3
Wiley
JS
Dubyak
GR
Extracellular adenosine triphosphate increases cation permeability of chronic lymphocytic leukemic lymphocytes.
Blood.
73
1989
1316
1323
4
Jamieson
GP
Snook
MB
Thurlow
PJ
Wiley
JS
Extracellular ATP causes loss of L-selectin from human lymphocytes via occupancy of P2Z purinoceptors.
J Cell Physiol.
166
1996
637
642
5
Wiley
JS
Gargett
CE
Zhang
W
Snook
MB
Jamieson
GP
Partial agonist and antagonist reveal a second permeability state of human lymphocyte P2Z/P2X7 channel.
Am J Physiol.
275
1998
C1224
C1231
6
Di Virgilio
F
Chiozzi
P
Ferrari
D
et al
Nucleotide receptors: an emerging family of regulatory molecules in blood cells.
Blood.
97
2001
587
600
7
Khakh
BS
Burnstock
G
Kennedy
C
et al
International Union of Pharmacology. XXIV. Current status of the nomenclature and properties of P2X receptors and their subunits.
Pharmacol Rev.
53
2001
107
118
8
Di Virgilio
F
Bronte
V
Collavo
D
Zanovello
P
Responses of mouse lymphocytes to extracellular adenosine 5′-triphosphate (ATP). Lymphocytes with cytotoxic activity are resistant to the permeabilizing effect of ATP.
J Immunol.
143
1989
1955
1960
9
Filippini
A
Taffs
RE
Agui
T
Sitkovsky
MV
EctoATPase activity in cytolytic T lymphocytes. Protection from the cytolytic effects of extracellular ATP.
J Biol Chem.
265
1990
334
340
10
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
1996
735
738
11
Baricordi
OR
Ferrari
D
Melchiorri
L
et al
An ATP-activated channel is involved in mitogenic stimulation of human T lymphocytes.
Blood.
87
1996
682
690
12
Perregaux
DG
McNiff
P
Laliberte
R
Conklyn
M
Gabel
CA
ATP acts as an agonist to promote stimulus-induced secretion of IL-12 and IL-18 in human blood.
J Immunol.
165
2000
4615
4623
13
Gu
BJ
Zhang
W
Worthington
RA
et al
A Glu-496 to Ala polymorphism leads to loss of function of the human P2X7 receptor.
J Biol Chem.
276
2001
11135
11142
14
Baricordi
OR
Melchiorri
L
Adinolfi
E
et al
Increased proliferation rate of lymphoid cells transfected with the P2X7 ATP receptor.
J Biol Chem.
274
1999
33206
33208
15
Bennett
JM
Catovsky
D
Daniel
MT
et al
Proposals for the classification of chronic (mature) B and T lymphoid leukemias. French-American-British (FAB) Cooperative Group.
J Clin Pathol.
42
1989
567
584
16
Chomczynski
P
Sacchi
N
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem.
162
1987
156
159
17
Ferrari
D
Munerati
M
Melchiorri
L
Hanau
S
Di Virgilio
F
Baricordi
OR
Responses to extracellular ATP of lymphoblastoid cell lines from Duchenne muscular dystrophy patients.
Am J Physiol.
267
1994
C886
C892
18
Markwardt
F
Lohn
M
Bohm
T
Klapperstuck
M
Purinoceptor-operated cationic channels in human B-lymphocytes.
J Physiol.
498
1997
143
151
19
Pizzo
P
Zanovello
P
Bronte
V
Di Virgilio
F
Extracellular ATP causes lysis of mouse thymocytes and activates a plasma membrane ion channel.
Biochem J.
274
1991
139
144
20
Gu
BG
Bendall
LJ
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
1998
946
951
21
Zanovello
P
Bronte
V
Rosato
A
Pizzo
P
Di Virgilio
F
Responses of mouse lymphocytes to extracellular ATP, II: extracellular ATP causes cell type-dependent lysis and DNA fragmentation.
J Immunol.
145
1990
1545
1550

Author notes

Francesco Di Virgilio, Dept of Experimental and Diagnostic Medicine, Section of General Pathology, Via Borsari, 46 I-44100 Ferrara, Italy; e-mail: fdv@dns.unife.it.

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