Doctor of Philosophy
School of Biological Sciences
Pupovac, Aleta, P2X7 receptor-induced CD23 shedding from B cells, Doctor of Philosophy thesis, School of Biological Sciences, University of Wollongong, 2014. http://ro.uow.edu.au/theses/4349
The P2X7 receptor is a ligand-gated cation channel, which is expressed on a variety of cell types, including human B cells. P2X7 activation induces a variety of downstream events, including the shedding of the immunoglobulin E receptor, CD23. Cell surface CD23 and soluble CD23 are important in the regulation of immunoglobulin E. Furthermore, soluble CD23 functions as a proinflammatory mediator. Thus it is important to elucidate the mechanisms involved in CD23 shedding. A disintegrin and metalloprotease (ADAM) 10 constitutively sheds CD23 from the surface of cells but whether P2X7 activates ADAM10, or other signalling processes to mediate CD23 shedding are unknown. Our laboratory has shown that P2X7 is expressed on human RPMI 8226 multiple myeloma B cells and that P2X7 activation on these cells induces the shedding of CD23. The primary aims of this thesis were: to confirm the presence of and to further characterise P2X7 in RPMI 8226 cells; to examine the signalling pathways involved in P2X7-induced CD23 shedding using this cell line as a model; to determine, using RPMI 8226 cells, whether ADAM10 is involved in P2X7-induced CD23 shedding; and finally to determine whether P2X7 activation induces CD23 shedding from primary human and murine B cells.
Messenger RNA (mRNA) expression of molecules was detected by reverse transcriptase-polymerase chain reaction. P2X7, CD23 and CXCL16 expression was detected by immunolabelling and measured by flow cytometry. Nucleotide-induced ethidium+ uptake (pore formation) was measured by flow cytometry or spectrofluorometry. Soluble CD23 and CXCL16 were measured by enzyme-linked immunosorbent assay. ATP-induced currents were measured by electrophysiology.
RPMI 8226 cells were shown to express mRNA for P2X7 and other P2X subtypes (P2X1, P2X4 and P2X5). Functional P2X7 was present on RPMI 8226 cells, and the new generation P2X7 antagonist AZ10606120, near-completely impaired both P2X7-induced pore formation and CD23 shedding in these cells. This data confirmed that the RPMI 8226 cell line is an adequate model to investigate the molecules and processes involved in P2X7-induced CD23 shedding.
Several signalling pathways involved in other P2X7-induced responses including reactive oxygen species formation, as well as changes in intracellular cation concentrations, were not involved in P2X7-induced CD23 shedding from RPMI 8226 cells. However, the phospholipase (PLD)1 antagonist, CAY10593 (VU0155069) impaired P2X7-induced CD23 shedding from RPMI 8226 cells. CAY10593 also impaired pore formation in RPMI 8226 cells, P2X7-transfected human embryonic kidney 293 cells and peripheral blood mononuclear cells. CAY10593 impaired P2X7-induced pore formation in RPMI 8226 cells more potently than the PLD2 antagonist CAY10594 and the non-specific PLD antagonist halopemide. CAY10593 also inhibited P2X7-mediated inward currents. Notably, PLD1 was absent in RPMI 8226 cells. This data indicates that CAY10593 impairs human P2X7 independently of PLD1 stimulation and highlights the importance of ensuring that compounds used in signalling studies downstream of P2X7 activation do not affect the receptor itself.
RPMI 8226 cells were shown to express mRNA for ADAM10. The ADAM10 antagonist, GI254023X significantly impaired P2X7-induced CD23 shedding from RPMI 8226 cells. ATP treatment of RPMI 8226 cells induced the rapid shedding of another ADAM10 substrate, CXCL16. The P2X7 antagonists, AZ10606120 and KN-62 near completely impaired ATP-induced CXCL16 shedding from RPMI 8226 cells and treatment of these cells with GI254023X significantly impaired P2X7-induced CXCL16 shedding. This data indicates that human P2X7 activation induces the rapid shedding of CD23 and CXCL16, and that these processes are mediated by ADAM10.
ATP treatment of primary human and murine B cells also induced the rapid shedding of CD23. Treatment of cells with AZ10606120, near-completely impaired ATP-induced CD23 shedding from both human and murine B cells. ATP-induced CD23 shedding was also impaired in B cells from P2X7 knockout mice. GI254023X impaired P2X7-induced CD23 shedding from both human and murine B cells. This data indicates that P2X7 activation induces the rapid shedding of CD23 from primary human and murine B cells, and that this process is also mediated by ADAM10.
Overall, this study shows, for the first time that ADAM10 mediates P2X7-induced CD23 and CXCL16 shedding from RPMI 8226 cells, as well as CD23 shedding from primary human and murine B cells. Moreover, this study excludes a potential role for various signalling molecules, including reactive oxygen species formation and the flux of various cations in P2X7-induced CD23 shedding. Finally, this study shows that the PLD1 antagonist, CAY10593, impairs P2X7 independently of PLD1.