Expression of P2 receptors in human B cells and Epstein-Barr virus-transformed lymphoblastoid cell lines
© Lee et al; licensee BioMed Central Ltd. 2006
Received: 29 April 2006
Accepted: 14 September 2006
Published: 14 September 2006
Epstein-Barr virus (EBV) infection immortalizes primary B cells in vitro and generates lymphoblastoid cell lines (LCLs), which are used for several purposes in immunological and genetic studies. Purinergic receptors, consisting of P2X and P2Y, are activated by extracellular nucleotides in most tissues and exert various physiological effects. In B cells, especially EBV-induced LCLs, their expression and function have not been well studied. We investigated the expression of P2 receptors on primary human B cells and LCLs using the quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method for revealing the gene expression profile of the P2 receptor subtypes and their changes during transformation.
The mRNA transcripts of most P2 receptors were detected in primary B cells; the expression of P2X3 and P2X7 receptors was the lowest of all the P2 receptors. By contrast, LCLs expressed several dominant P2 receptors – P2X4, P2X5, and P2Y11 – in amounts similar to those seen in B cells infected with EBV for 2 weeks. The amount of most P2 subtypes in LCLs or EBV-infected B cells was lower than in normal B cells. However, the amount of P2X7 receptor expressed in LCLs was higher. Protein expression was studied using Western blotting to confirm the mRNA findings for P2X1, P2X4, P2X7, P2Y1, and P2Y11 receptors. ATP increased the intracellular free Ca2+ concentration ([Ca2+]i) by enhancing the Ca2+ influx in both B cells and LCLs in a dose-dependent manner.
These findings describe P2 receptor expression profiles and the effects of purinergic stimuli on B cells and suggest some plasticity in the expression of the P2 receptor phenotype. This may help explain the nature and effect of P2 receptors on B cells and their role in altering the characteristics of LCLs.
B cells synthesize and secrete large quantities of soluble immunoglobulin antibodies and thus, play a key role in humoral immunity. An infection with the Epstein-Barr virus (EBV) easily transforms resting primary B cells in vitro from human peripheral blood cells into B-blast-like proliferating lymphoblastoid cell lines (LCLs) . This infection is used routinely in the laboratory to generate LCLs from B cells . LCLs are widely used in various types of studies, including those involving the disciplines of immunology, cellular biology, and genetics. This transformation results in changes in certain cellular properties, including gene expression , cell surface phenotyping, and cytokine production .
Extracellular nucleotides – e.g., adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate, uracil 5'-triphosphate, and uracil 5'-diphosphate – have various physiological effects in many cells, such as exocrine and endocrine secretion, neurotransmission, cell proliferation, cell differentiation, and programmed cell death that are mediated by P2 receptors, consisting of P2X and P2Y receptors . P2X receptors are ligand-gated cation channels, of which seven receptor subtypes (P2X1 to P2X7) have been identified and cloned . P2Y receptors, which are G-protein-coupled metabotropic structures, consist of eight cloned and functionally distinct subtypes: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14 [5, 7].
Blood cells express P2 receptors which regulate such responses as cell proliferation, differentiation, chemotaxis, cytokine release, immune and inflammatory responses [5, 8]. In lymphocytes, ATP induces an increase in membrane permeability for cations and larger molecules [9, 10], as well as cellular proliferation  and cell death through P2 receptors [12, 13]. The precise nature of the expression and function of the P2 receptor subtypes have been investigated [14–16].
P2 receptors expressed in B cells have been investigated using electrophysiological, pharmacological, and immunocytochemical techniques, which have revealed the existence of P2 receptors , especially P2X [14, 18]. However, the researchers in these studies failed to perform a quantitative analysis of P2 mRNA and used B cells from chronic lymphocytic leukemia (CLL) or LCLs, rather than pure B cells. Recently, the mRNA profile of the lymphocyte P2 receptor was subjected to quantitative analysis, but the B cells were not separated and not all subtypes were targeted [15, 16].
In this study, we investigated the expression of P2 receptors in human B cells and in LCLs using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and fluorimetric techniques to measure intracellular free Ca2+ concentration ([Ca2+]i). We were able to determine the profile of the P2 receptor mRNA in these cells and monitor changes in [Ca2+]i in response to P2 receptor activation. Our findings indicate the plasticity of P2 receptors in B cells during their transformation into LCLs.
IgD and CD38 are cell-surface molecules that have been used widely to identify the B-cell phenotype during B-cell development. Like germinal center B cells, most EBV-transformed B cells were positive for CD38 but not for IgD [19, 20]. The expression of IgD and CD38 molecules on primary B cells and EBV-transformed LCLs was evaluated by fluorescence-activated cell sorter (FACS) analysis. To generate LCLs, we cultured isolated B cells with the active EBV supernatant for 4 to 6 weeks, as described in Methods section. The primary B cells expressed IgD, but not CD38, and the LCLs expressed CD38, but not IgD (data not shown). This result is consistent with our previous findings .
P2 receptor mRNA quantification
Western blotting for P2 receptors
Effect of ATP on intracellular free Ca2+ concentration
In this study, we determined and compared mRNA expression levels for all known P2X and P2Y receptor subtypes on human B cells and LCLs. Quantitative RT-PCR was used to determine the gene expression profile for P2 receptors. This method was selected because selective agonists and antagonists for most of the P2 receptor subtypes are absent and real-time PCR has advantages over other methods, such as requiring only a small number of cells and being one of the most reliable methods of determining the amount of RNA.
This is the first study to show the expression of P2 receptors using mRNA from healthy human B cells. In these cells, most of the P2X and P2Y receptor subtypes had 2-fold expression with the exception of P2X3 and P2X7 receptors. In the studies of the P2X receptor, the P2X1, P2X2, P2X4, and P2X7 receptors were found in human B cells by an immunocytochemical assay  and the non-desensitizing cation channels activated by ATP, which is a feature of P2X7 receptor, were measured using electrophysiological methods . The different results of P2X subtype expression might be due to the different B cells, or variations in P2X receptor expression . B cells transformed by EBV  or malignant B cells  were used in previous studies, while normal B cells were used in the present study. In addition, it is possible that there might be differences in the transcription, translation, and function of P2X receptors. The different P2X7 expression levels may be because P2X7 receptor might be up-regulated in CLLs  and that some lymphoid cells do not express P2X7 receptor . In addition, B cells did not undergo the typical increase in membrane permeability to ATP and were not susceptible to ATP-mediated cytotoxicity [8, 22]. Although the P2Y receptors in B cells were investigated, it was not enough to compare the expression of subtypes. P2Y subtypes were detected by RT-PCR in previous studies, albeit only in lymphocytes [15, 16].
In LCLs and B cells infected by EBV for 2 weeks, the predominant P2 receptor subtypes were P2X1, P2X4, P2X5, P2X7, and P2Y11. The expression of most P2 receptors was suppressed during the EBV-induced B-cell transformation into LCLs, however, the suppression of P2X1, P2X4, P2X5 and P2Y11 receptors was not as great as for other subtypes. Only P2X7 receptor was significantly up-regulated. Western blotting showed similar patterns for P2X1, P2X4, P2X7, P2Y1, and P2Y11, as well as for P2X2, P2X5, P2Y2, and P2Y6 (data not shown). Our results suggest that there is some plasticity in P2-receptor expression in B cells. This possibility has been investigated in many tissues and cells, including the urinary bladder, heart, vessels, gut, neurons, and cancer cells . In immune cells, plasticity in P2Y2-receptor expression was studied during myeloid leukocyte differentiation . Sensitivity to ATP in thymocytes changes with the stage of maturation [24, 25], and P2X7-receptor expression can be modulated by diverse stimuli . The plasticity of P2 receptors may be due to changes in their exposure to ATP or EBV-induced changes in gene expression. In vivo, ATP is often released by blood cells into the extracellular environment through nonlytic mechanisms. Some leakage of cytoplasmic ATP may also occur as a consequence of damage to the cell or acute cell death. Platelet-dense granules comprise another relevant source of ATP . In vitro, however, the sources of ATP for B cells are limited to nonlytic mechanisms or leakage of cytoplasmic ATP. The EBV-induced transformation of B cells into LCLs results in some B cells dying, which results in ATP being released into the extracellular compartment, where it continually degrades. Thus, the concentration of ATP may be high in the early stages of in vitro transformation and lower in later stages. The expression of P2 receptors may be affected by this fluctuation in environmental ATP.
In PBMC populations that include lymphocytes and monocytes, the dominant P2 receptor subtypes were P2X4, P2Y6, P2Y11, and P2Y13. An mRNA expression assay revealed that the P2Y1, P2Y2, P2Y4, and P2Y6 receptors were expressed in lymphocytes and monocytes and that the P2Y6 receptor was expressed in relatively higher amounts than the other P2Y receptor subtypes . P2X4 and P2Y12 receptors were expressed in relatively large amounts in lymphocytes and P2X4, P2Y2, and P2Y13 receptors in monocytes . The expression of P2X4, P2Y6, and P2Y13 receptors correlated with the findings of previous studies; however, the expression of the P2Y11 receptor was somewhat different. It is possible that other lymphocytes or monocytes expressed these subtypes predominantly. Alternatively this may reflect a variation in cohorts or contamination with other types of blood cells. To date, these blood cells have not been investigated well enough to compare P2 receptor subtypes, although some of them have been surveyed [5, 8, 16, 27, 28]. Because the P2 receptor profiles of blood cells are not completely known, it is difficult to determine which P2 receptor subtypes have been expressed dominantly in PBMCs until now.
Although the P2Y8 and P2Y10 receptors were examined with other subtypes, the findings for these subtypes were omitted because they are not included among the classical P2Y receptor subtypes in humans. We found the mRNA for these subtypes in B cells, LCLs, and PBMCs, indicating that they are prominent in these cells. In previous studies of human P2 receptors, the P2Y8 and P2Y10 receptors were expressed in HL60  and included in the human genome . The National Center for Biotechnology Information (NCBI) confirmed the gene sequence for each of these receptors (P2Y8; NM_178129, P2Y10; NM_014499). A physiological role for the P2Y8 and P2Y10 receptors in B cells and in human blood cells can therefore be expected.
ATP-stimulated P2 receptors increased the [Ca2+]i in B cells and LCLs, albeit rather slowly. This was quite a different effect from that of other stimuli, such as the anti-IgM antibody, which caused [Ca2+]i levels to change rapidly . This might be due to differences in Ca2+ signaling or the temperature at which the experiments were conducted, which might influence the kinetics involved when the [Ca2+]i changes. Extracellular Ca2+-free conditions prevented the [Ca2+]i from increasing, thereby indicating that the main cause of the increase in [Ca2+]i might be an ATP-induced influx of Ca2+, although the possibility that mobilization of stored Ca2+ may be involved should probably also be considered. The increased [Ca2+]i might be largely due to P2X receptor activity, because it was mediated by an influx of Ca2+, which is the major effect of activating P2X receptors. The results of real-time PCR indicated a decrease in P2 receptors and those of Western blotting demonstrated a similar pattern for several P2 receptors, even when the up-regulation of P2X7 receptor was considered. However, the increase in [Ca2+]i by ATP was a little higher in LCLs than in B cells, which was not statistically significant. EBV-transformed B cells might enhance the availability of Ca2+, thereby causing [Ca2+]i to rise, even when P2 receptors are down-regulated. The response to extracellular ATP is an increase in [Ca2+]i in B cells as well as in LCLs, which are probably derived from EBV-infected B cell lymphoma in vivo. This may cause a variety of cellular events, ranging from transcriptional regulation to cell migration and proliferation.
In this study, the expression of P2X and P2Y receptors in human B cells and LCLs was investigated. P2-receptor expression was suppressed during the EBV-induced transformation of B cells, except for the P2X7 subtype, which was up-regulated. Extracellular ATP induced an increase in [Ca2+]i in B cells and LCLs via P2 receptors. Therefore, these findings reveal the exact P2 receptor profiles and the effects of purinergic stimuli on B cells and suggest some plasticity in the expression of the P2 receptor phenotype. This will help us explain the nature and effect of P2 receptors on B cells and their role in altering the characteristics of LCLs.
B-cell purification and generation of EBV-transformed LCLs
Ten 240-mL packs of blood were obtained from the Central Red Cross Blood Center (Seoul, Korea). This blood was not appropriate for transfusion because of slightly elevated alanine aminotransferase levels. We used it to isolate PBMCs, using Ficoll-Hypaque gradient centrifugation (Amersham Biosciences, Uppsala, Sweden) and B cells, which were purified (>95% CD20+) using a B-cell isolation kit and a MACS separator (Miltenyi Biotec, Bergisch Gladbach, Germany). The immortalization of B cells was achieved by EBV infection [2, 32–34]. The B95-8 supernatant was added to the purified B cells in a culture flask (1 × 106 cells/mL). Following a 2-hour incubation period at 37°C, the same volume of medium and 0.5 μg/mL cyclosporine A  were added. The cultures were incubated for 4 to 6 weeks until clumps of EBV-infected B cells were visible. EBV-transformed LCLs were cultured in RPMI-1640 medium (GIBCO/BRL, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (BioWhittaker, Walkerville, MD, USA) and 1% (v/v) antibiotics/antimycotics that included penicillin G (100 IU/mL), streptomycin (100 μg/mL), and amphotericin B (0.25 μg/mL). The cells were cultured in a humidified atmosphere of 5% CO2 and 95% air at 37°C. The EBV stock was prepared from an EBV-transformed B95-8 marmoset cell line. These cells were grown in an RPMI-1640 medium supplemented with 10% FBS, and infectious culture supernatants were harvested and stored at -80°C until needed. Thus, each pack of blood was used to produce B cells, EBV-infected B cells, LCLs, and PBMCs for use in this experiment. The study was approved by the Institutional Review Board at the National Institute of Health, Korea Center for Disease Control and Prevention.
Quantitative real-time RT-PCR
Sequence details for all P2X and P2Y receptor subtypes and reference (GAPDH) primers.
471 – 491
538 – 514
958 – 975
1024 – 1005
135 – 152
205 – 182
1108 – 1128
1176 – 1155
311 – 328
378 – 360
488 – 507
555 – 536
401 – 425
476 – 458
1352 – 1370
1419 – 1399
1495 – 1520
1567 – 1551
725 – 742
793 – 776
1171 – 1186
1227 – 1209
511 – 530
586 – 567
318 – 339
385 – 368
223 – 248
291 – 274
433 – 456
505 – 486
227 – 244
295 – 274
Cells were lysed in RIPA buffer containing 150 mM NaCl, 50 mM Tris-Cl (pH 7.2), 1% sodium deoxycholate, 0.1% SDS, 1 μg/mL aprotinin, 1 mM EGTA, 1 mM PMSF, and 1 mM sodium orthovanadate. After incubation in ice for 20 minutes on a shaking platform, the samples were centrifuged at 10,000 × g for 5 minutes at 4°C. Proteins were mixed with the sample buffer (50 mM Tris-Cl, pH 6.8, 10% glycerol, 2% SDS, 1% mercaptoethanol, and 0.1% bromophenol blue), heated to 95°C for 5 minutes, and separated on a 10% SDS-PAGE gel. The gel was transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Buckingshire, UK) and blocked in TBST (20 mM Tris-Cl, pH 7.6, 137 mM NaCl, 2.7 mM KCl, and 0.1% Tween 20) containing 5% (v/v) nonfat milk powder for 2 hours at room temperature. The membrane was incubated with rabbit polyclonal antibodies (Alomone Labs, Jerusalem, Israel) against P2X1 receptor, P2X4 receptor, P2X7 receptor, P2Y1 receptor, or P2Y11 receptor in TBST for 2 hours at room temperature, washed with TBST, and incubated with secondary anti-rabbit IgG (Amersham Pharmacia Biotech) in TBST for 1 hour. After the membrane was washed in TBST, protein bands were visualized using Western Lightning (PerkinElmer Life Sciences Inc., Gaithersburg, MD, USA). To compare protein loading, the blot was re-probed with anti-GAPDH antibody (Novus Biologicals, Littleton, CO, USA).
Intracellular Ca2+ measurements
The [Ca2+]i was measured using a single-cell microscopy technique with Fura 2 [31, 37]. B cells and LCLs were suspended in culture and allowed to attach to glass coverslips coated with Poly-L-lysine (100 μg/mL; Sigma-Aldrich, St. Louis, MO, USA) and incubated for at least 3 hours before use. The cells were loaded with the cell-permeable Ca2+ indicator Fura 2-AM (5.0 μM; Molecular Probes, Eugene, OR, USA) in the culture medium for 1 hour at room temperature and then washed and bathed in an external solution (135 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES at pH 7.4) for at least 20 minutes before Ca2+ measurements were made. Glass coverslips were placed into a chamber (Warner Instrument, Hamden, CT, USA) on an inverted microscope (Olympus, Tokyo, Japan), and the fluorescence intensities of the Fura-2-loaded cells were measured using a digital fluorescence imaging system. Discrete bandwidth excitation light (340 nm, 380 nm) was delivered to the epifluorescence attachment of the microscope through a quartz fiber-optic guide. The fluorescence emitted by the Fura-2-loaded cells was passed through a 510-nm-long pass filter, and images were obtained using a cooled charge-coupled device camera (Roper Scientific, Trenton, NJ, USA). Fluorescent video images were averaged, digitized (0.3–1.0 Hz), and analyzed using Metafluor acquisition and analysis software (Universal Imaging Corp, West Chester, PA, USA). Individual cells in the field of view were selected and paired 340/380 images were subtracted from the background. The Fura-2 fluorescence ratios, indicative of changes in [Ca2+]i, were calculated and their changes were extracted over time. All experiments were performed at room temperature, and the external solution and drugs were perfused at a rate of 2 mL/min by gravity. Data were expressed as the ratio of fluorescence due to excitation at 340 nm and at 380 nm (F340:380). In some experiments, a nominally Ca2+-free medium was used, which was identical in composition, except for the omission of CaCl2.
Data are presented as the mean ± SEM, and n indicates the number of independent experiments or the number of cells used to measure [Ca2+]i. Statistical significance was determined using one-way ANOVA or Student's t test; p < 0.05 was considered significant.
This work was supported by an intramural grant (2005-N-00179-00) from the Center for Genome Sciences, National Institute of Health, Korea Center for Disease Control and Prevention, Seoul, South Korea.
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