P2X₇ receptor expression in evolutive and indolent forms of chronic B 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.

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.

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

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).

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.

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.

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.

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).

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.