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

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). P2X₁ or P2Y₁ were used as a calibrator (i.e. the P2X receptor was expressed as a ratio of P2X₁ and the P2Y receptor as P2Y₁) 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 2-fold of each other with the exception of the P2X₃ and P2X₇ receptors, which were expressed in lower quantities (Figure 1, n = 4). P2X₇ receptor expression was significantly low compared to other P2X receptors (p < 0.05), with the exception of P2X₃. 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]. EBV-infected B cells were also examined because an in vitro transformation might alter the expression of receptors. The most abundant P2 receptor subtypes were P2X₄, P2X₅, and P2Y₁₁ (Figure 2; n = 4). The expression of P2X₅ 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, P2X₁ of B cells used as a P2X calibrator and P2Y₁ as a P2Y calibrator). The expression of the P2X₁ through to P2X₆ 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 P2X₇ 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, P2X₄, P2Y₆, P2Y₁₁, and P2Y₁₃ were the predominant P2 receptor subtypes (Figure 4, n = 4), and the expression rates for P2X₄ and P2Y₆ were significantly higher than other P2X or P2Y subtypes (p < 0.001). In addition, P2X₄, P2X₇, P2Y₆, P2Y₁₁, and P2Y₁₃ 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 P2X₇ receptor can be expected to have a physiological role during the transformation of B cells into LCLs.

To investigate the correlation of mRNA with protein, we carried out Western blot analysis for P2X₁, P2X₄, P2X₇, P2Y₁, and P2Y₁₁ 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 P2X₁ (60-kDa), P2X₄ (65-kDa), P2Y₁ (66-kDa), and P2Y₁₁ (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 P2X₇ 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 P2X₇ is higher in LCLs than in B cells. To compare protein loading, the blot was re-probed with anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (40-kDa) (Figure 5, right).

Extracellular ATP is an effective modulator of [Ca²⁺]_i, and its activities are mediated through P2 receptors [17]. To determine whether the P2 receptors examined by RT-PCR and Western blotting were functional, we carried out digital fluorescence imaging of the Ca²⁺-sensitive dye Fura 2-AM and compared the images seen following ATP-induced changes in [Ca²⁺]_i in B cells and LCLs. In the presence of 2 mM Ca²⁺ and 1 mM ATP, we observed an average increase in [Ca²⁺]_i from 0.1 to 0.5 (representing the F340:F380 ratio) in B cells and LCLs (Δratio of B cells: 0.32 ± 0.04, n = 6; Δratio of LCLs: 0.39 ± 0.04, n = 6) (Figures 6A and 6B). The [Ca²⁺]_i increased with ATP in a dose-dependent manner (100–1000 μM; n = 25) (Figure 6C), indicating that the potency of ATP was similar in both cells. The mechanism leading to the intracellular Ca²⁺ response was examined further by repeating these experiments under Ca²⁺-free conditions. The B cells were treated with 1 mM ATP under Ca²⁺-free conditions, and the [Ca²⁺]_i remained at or near the pre-agonist levels (Figure 7A). The peak [Ca²⁺]_i was mostly abolished in B cells (n = 14, p < 0.0001) and in LCLs (n = 12, p < 0.0001) in the absence of Ca²⁺ (Figure 7B). These data suggest that an influx of Ca²⁺ is the major route by which B cells and LCLs respond to ATP stimulation.

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 [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% 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.

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 oligodT₁₅ (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 efficiency 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 P2X₁ or P2Y₁ as a calibrator and applying the formula 2^-ΔΔCt[36].

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).

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.