Expression of P2 receptors in human B cells and Epstein-Barr virus-transformed lymphoblastoid cell lines

Background 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. Results 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. Conclusion 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.


Background
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) [1]. This infection is used routinely in the laboratory to generate LCLs from B cells [2]. 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 [3], cell surface phenotyping, and cytokine production [4]. 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 [5]. P2X receptors are ligand-gated cation channels, of which seven receptor subtypes (P2X 1 to P2X 7 ) have been identified and cloned [6]. P2Y receptors, which are G-protein-coupled metabotropic structures, consist of eight cloned and functionally distinct subtypes: P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , P2Y 13 , and P2Y 14 [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 [11] 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][15][16].
P2 receptors expressed in B cells have been investigated using electrophysiological, pharmacological, and immunocytochemical techniques, which have revealed the existence of P2 receptors [17], 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 Ca 2+ concentration ([Ca 2+ ] i ). We were able to determine the profile of the P2 receptor mRNA in these cells and monitor changes in [Ca 2+ ] i in response to P2 receptor activation. Our findings indicate the plasticity of P2 receptors in B cells during their transformation into LCLs.

Results
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 EBVtransformed 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 [20].

P2 receptor mRNA quantification
The expression of P2 receptors in B cells and LCLs was determined using quantitative RT-PCR. The expression of the P2 receptor subtypes was compared among B cells, LCLs, and peripheral blood mononuclear cells (PBMCs). P2X 1 or P2Y 1 were used as a calibrator (i.e. the P2X receptor was expressed as a ratio of P2X 1 and the P2Y receptor as P2Y 1 ) in order to illustrate the expression of P2 receptors relative to each other. All P2X and P2Y receptor subtypes were detected in the B cells. Most of the P2 receptor subtypes had similar rates of expression within 1-or 2fold of each other with the exception of the P2X 3 and P2X 7 receptors, which were expressed in lower quantities (Figure 1, n = 4). P2X 7 receptor expression was significantly low compared to other P2X receptors (p < 0.05), with the exception of P2X 3 . The presence of P2X-and P2Y-receptor mRNA in the B cells is in agreement with the findings of previous lymphocyte studies using RT-PCR [15,16]. EBVinfected B cells were also examined because an in vitro transformation might alter the expression of receptors. The most abundant P2 receptor subtypes were P2X 4 , P2X 5 , and P2Y 11 ( Figure 2; n = 4). The expression of P2X 5 receptors in LCLs was significantly higher than other P2X receptors (p < 0.05; Figure 2). The P2 receptors in B cells were compared with those expressed in LCLs ( Figure 3, P2X 1 of B cells used as a P2X calibrator and P2Y 1 as a P2Y calibrator). The expression of the P2X 1 through to P2X 6 receptors and P2Y receptors in LCLs and B cells that had been infected with EBV for 2 weeks was significantly lower than in noninfected B cells (p < 0.01; Figure 3). However, the LCLs expressed a significantly larger number of P2X 7 receptors than B cells (p < 0.01; Figure 3). The expression of EBV-infected LCLs, which had been infected for more than 4 weeks and EBV-infected B cells, which had been infected for 2 weeks, yielded similar profiles and quantities. As a control, P2 receptors in PBMCs were quantified and these showed a different expression profile. In PBMCs, which are mainly monocytes and lymphocytes, P2X 4 , P2Y 6 , P2Y 11 , and P2Y 13 were the predominant P2 receptor subtypes (Figure 4, n = 4), and the expression rates for P2X 4 and P2Y 6 were significantly higher than other P2X or P2Y subtypes (p < 0.001). In addition, P2X 4 , P2X 7 , P2Y 6 , P2Y 11 , and P2Y 13 expression was significantly higher in PBMCs compared with B cells (p < 0.05), which may be the result of T cell/monocyte contamination in the PBMC preparation [15,16]. Therefore, the up-regulated P2X 7 receptor can be expected to have a physiological role during the transformation of B cells into LCLs.

Western blotting for P2 receptors
To investigate the correlation of mRNA with protein, we carried out Western blot analysis for P2X 1 , P2X 4 , P2X 7 , P2Y 1 , and P2Y 11 receptors, all of which had varying amounts of mRNA during EBV transformation (n = 4). The distribution of P2 receptors in B cells and LCLs is shown on the left panel ( Figure 5). The bands representing P2X 1 (60-kDa), P2X 4 (65-kDa), P2Y 1 (66-kDa), and P2Y 11 (50-kDa) receptors were more prominent in B cells than in LCLs, which correlates with the results of the mRNA quantitative analysis. As for the P2X 7 receptor, it was represented by a prominent 68-kDa band in LCLs and a faint band in B cells. This is consistent with the results of RT-PCR, which indicated that the expression of P2X 7 is higher in LCLs than in B cells. To compare protein loading, the blot was re-probed with anti-glyceraldehyde-3phosphate dehydrogenase (GAPDH) antibody (40-kDa) ( Figure 5, right).

Effect of ATP on intracellular free Ca 2+ concentration
Extracellular ATP is an effective modulator of [Ca 2+ ] i , and its activities are mediated through P2 receptors [17]. To determine whether the P2 receptors examined by RT-PCR

Ratio of Target/P2Y1
Relative expression of P2 receptors in B cells Figure 1 Relative expression of P2 receptors in B cells. The relative expression of the P2X receptor gene (upper; P2X 1 , P2X 2 , P2X 3 , P2X 4 , P2X 5 , P2X 6 , and P2X 7 ) and P2Y receptor gene (lower; P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , P2Y 13 , and P2Y 14 ) in B cells are presented (n = 4). The expression was normalized to GAPDH. P2X receptors were calibrated by P2X 1 and P2Y receptors were calibrated by P2Y 1 . Data are the mean ± SEM.  Figure 6C), indicating that the potency of ATP was similar in both cells. The mechanism leading to the intracellular Ca 2+ response was examined further by repeating these experiments under Ca 2+ -free conditions. The B cells were treated with 1 mM ATP under Ca 2+ -free conditions, and the [Ca 2+ ] i remained at or near the pre-agonist levels ( Figure  7A). The peak [Ca 2+ ] i was mostly abolished in B cells (n = 14, p < 0.0001) and in LCLs (n = 12, p < 0.0001) in the absence of Ca 2+ ( Figure 7B). These data suggest that an influx of Ca 2+ is the major route by which B cells and LCLs respond to ATP stimulation.

Discussion
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 P2X 3 and P2X 7 receptors. In the studies of the P2X receptor, the P2X 1 , P2X 2 , P2X 4 , and P2X 7 receptors were found in human B cells by an immunocytochemical assay [14] and the nondesensitizing cation channels activated by ATP, which is a feature of P2X 7 receptor, were measured using electrophysiological methods [18]. The different results of P2X subtype expression might be due to the different B cells, or variations in P2X receptor expression [21]. B cells transformed by EBV [18] or malignant B cells [14] 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 P2X 7 expression levels may be because P2X 7 receptor might be up-regulated in CLLs [10] and that some lymphoid cells do not express P2 receptors (P2X 1 , P2X 4 , P2X 7 , P2Y 1 , and P2Y 11 ) compared by Western blotting in B cells and LCLs    [11]. 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].

Relative expression of P2 receptors in PBMCs
In LCLs and B cells infected by EBV for 2 weeks, the predominant P2 receptor subtypes were P2X 1 , P2X 4 , P2X 5 , P2X 7 , and P2Y 11 . The expression of most P2 receptors was suppressed during the EBV-induced B-cell transformation into LCLs, however, the suppression of P2X 1 , P2X 4 , P2X 5 and P2Y 11 receptors was not as great as for other subtypes.  Only P2X 7 receptor was significantly up-regulated. Western blotting showed similar patterns for P2X 1 , P2X 4 , P2X 7 , P2Y 1 , and P2Y 11 , as well as for P2X 2 , P2X 5 , P2Y 2 , and P2Y 6 (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 [5]. In immune cells, plasticity in P2Y 2receptor expression was studied during myeloid leukocyte differentiation [23]. Sensitivity to ATP in thymocytes changes with the stage of maturation [24,25], and P2X 7receptor expression can be modulated by diverse stimuli [26]. 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 [8]. 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 P2X 4 , P2Y 6 , P2Y 11 , and P2Y 13 . An mRNA expression assay revealed that the P2Y 1 , P2Y 2 , P2Y 4 , and P2Y 6 receptors were expressed in lymphocytes and monocytes and that the P2Y 6 receptor was expressed in relatively higher amounts than the other P2Y receptor subtypes [16]. P2X 4 and P2Y 12 receptors were expressed in relatively large amounts in lymphocytes and P2X 4 , P2Y 2 , and P2Y 13 receptors in monocytes [15]. The expression of P2X 4 , P2Y 6 , and P2Y 13 receptors correlated with the findings of previous studies; however, the expression of the P2Y 11 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 P2Y 8 and P2Y 10 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 P2Y 8 and P2Y 10 receptors were expressed in HL60 [29] and included in the human genome [30].

Conclusion
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 P2X 7 subtype, which was up-regulated. Extracellular ATP induced an increase in [Ca 2+ ] 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][33][34]. The B95-8 supernatant was added to the purified B cells in a culture flask (1 × 10 6 cells/mL). Following a 2-hour incubation period at 37°C, the same volume of medium and 0.5 μg/mL cyclosporine A [35] 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% CO 2 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
The total cellular RNA was collected from human B cells, LCLs, and PBMCs. RNA was extracted using the RNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions and stored at -80°C until used. Quantitative RT-PCR was performed to determine the expression of the P2 receptor genes (Table 1). To generate cDNA, we induced reverse transcription of the total RNA using oligodT 15 (Roche Diagnostics GmbH, Mannheim, Germany) and reverse transcription polymerase (Promega, Madison, WI, USA). Oligonucleotide primers (Bioneer, Daejeon, Korea) were designed using Primer Express software (Applied Biosystems, Foster City, CA, USA), based on sequences obtained from the GenBank database, and tested for quality and efficiency. Primer effi-ciency was established to ensure optimal amplification of our samples. Serial dilutions of synthetic cDNAs were carried out according to the supplier's instructions to define relative changes in quantity. Real-time PCR was performed using SYBR Green PCR Master mix (Applied Biosystems) in an ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems). The amplification program included activation of AmpliTaq Gold at 95°C for 10 minutes, followed by 45 cycles of 2-step PCR with denaturation at 95°C for 15 seconds and annealing/extension at 60°C for 1 minute. Amplifications were followed by a melting curve analysis. A negative control (no cDNA template) was run simultaneously with every assay. The PCR from each cDNA sample was run in triplicate. Constitutively expressed GAPDH was selected as an endogenous control to correct any potential variation in RNA loading or in the efficiency of the amplification reaction. Results are presented as relative fold changes by using GAPDH as a reference and P2X 1 or P2Y 1 as a calibrator and applying the formula 2 -ΔΔCt [36].  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 [Ca 2+ ] 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 Ca 2+ -free medium was used, which was identical in composition, except for the omission of CaCl 2 .

Statistics
Data are presented as the mean ± SEM, and n indicates the number of independent experiments or the number of cells used to measure [Ca 2+ ] i . Statistical significance was determined using one-way ANOVA or Student's t test; p < 0.05 was considered significant.