Abstract
BackgroundEpstein-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.ResultsThe 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.ConclusionThese 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.
Highlights
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
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)
The expression of IgD and CD38 molecules on primary B cells and EBV-transformed LCLs was evaluated by fluorescence-activated cell sorter (FACS) analysis
Summary
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. BMC Immunology 2006, 7:22 http://www.biomedcentral.com/1471-2172/7/22 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]. 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]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
