P2X7 receptor-induced CD23 shedding from B cells

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

Human P2X7 receptor activation induces the rapid shedding of CXCL16

Activation of the purinergic P2X7 receptor by extracellular ATP induces the shedding of cell-surface molecules including the low-affinity IgE receptor, CD23 from leukocytes. CD23 is a known substrate of a disintegrin and metalloprotease (ADAM)10. The aim of the current study was to determine if P2X7 activation induced the shedding of the chemokine CXCL16, an ADAM10 substrate. Using immunolabelling and flow cytometry we demonstrate that human RPMI 8226 multiple myeloma B cells, which have been previously shown to express P2X7, also express CXCL16. Flow cytometric and ELISA measurements of ATP-induced loss of cell-surface CXCL16 showed that ATP treatment of RPMI 8226 cells induced the rapid shedding of CXCL16. Treatment of RPMI 8226 cells with the specific P2X7 antagonists, AZ10606120 and KN-62 impaired ATP-induced CXCL16 shedding by ∼86% and ∼90% respectively. RT-PCR demonstrated that ADAM10 is expressed in these cells and treatment of cells with the ADAM10 inhibitor, GI254023X, impaired ATP-induced CXCL16 shedding by ∼87%. GI254023X also impaired P2X7-induced CD23 shedding by ∼57%. This data indicates that human P2X7 activation induces the rapid shedding of CXCL16 and that this process involves ADAM10

Activation of the P2X7 receptor induces the rapid shedding of CD23 from human and murine B cells

Activation of the P2X7 receptor by the extracellular damage-associated molecular pattern, adenosine 5′-triphosphate (ATP), induces the shedding of cell surface molecules including the low-affinity IgE receptor, CD23, from human leukocytes. A disintegrin and metalloprotease (ADAM) 10 mediates P2X7-induced shedding of CD23 from multiple myeloma RPMI 8226 B cells; however, whether this process occurs in primary B cells is unknown. The aim of the current study was to determine whether P2X7 activation induces the rapid shedding of CD23 from primary human and murine B cells. Flow cytometric and ELISA measurements showed that ATP treatment of human and murine B cells induced the rapid shedding of CD23. Treatment of cells with the specific P2X7 antagonist, 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. The absence of full-length, functional P2X7 in the P2X7 knockout mice was confirmed by immunoblotting of splenic cells, and by flow cytometric measurements of ATP-induced YO-PRO-12+ uptake into splenic B and T cells. The broad-spectrum metalloprotease antagonist, BB-94, and the ADAM10 antagonist, GI254023X, impaired P2X7-induced CD23 shedding from both human and murine B cells. These data indicate that P2X7 activation induces the rapid shedding of CD23 from primary human and murine B cells and that this process may be mediated by ADAM10

P2X7 receptor activation mediates organic cation uptake into human myeloid leukaemic KG-1 cells

The P2X7 purinergic receptor is an ATP-gated cation channel with an emerging role in neoplasia. In this study we demonstrate that the human KG-1 cell line, a model of acute myelogenous leukaemia, expresses functional P2X7. RT-PCR and immunochemical techniques demonstrated the presence of P2X7 mRNA and protein respectively in KG-l cells, as well as in positive control multiple myeloma RPMI 8226 cells. Flow cytometric measurements demonstrated that ATP induced ethidium(+) uptake into KG-l cells suspended in sucrose medium (EC(50) of ∼3 μM), but not into cells in NaCl medium. In contrast, ATP induced ethidium(+) uptake into RPMI 8226 cells suspended in either sucrose or NaCl medium (EC(50) of ∼3 or ∼99 μM, respectively), as well as into RPMI 8226 cells in KCl medium (EC(50) of ∼18 μM). BzATP and to a lesser extent ATPγS and αβ-methylene ATP, but not ADP or UTP, also induced ethidium(+) uptake into KG-1 cells. ATP-induced ethidium(+) uptake was completely impaired by the P2X7 antagonists, AZ10606120 and A-438079. ATP-induced ethidium(+) uptake was also impaired by probenecid but not by carbenoxolone, both pannexin-1 antagonists. ATP induced YO-PRO-1(2+) and propidium(2+) uptake into KG-1 cells. Finally, sequencing of full-length P2X7 cDNA identified several single nucleotide polymorphisms (SNPs) in KG-1 cells including H155Y, A348T, T357S and Q460R. RPMI 8226 cells contained A348T, A433V and H521Q SNPs. In conclusion, the KG-1 cell line expresses functional P2X7. This cell line may help elucidate the signalling pathways involved in P2X7-induced survival and invasiveness of myeloid leukaemic cells

Human P2X7 receptor activation induces ADAM10-mediated shedding of CD23 and CXCL16 (P4137)

Activation of the damage-associated molecular pattern receptor P2X7 by extracellular ATP induces the shedding of cell surface molecules, including CD23, from leukocytes. The mechanisms involved in this process however remain poorly understood. Using a flow cytometric assay, we demonstrated that ATP-induces the rapid shedding of CD23 from the surface of human multiple myeloma RPMI 8226 and peripheral blood B cells with a t1/2 of 7 min. The P2X7 antagonist, AZ10606120, impaired ATP-induced CD23 shedding by 89%. Moreover, P2X7-induced CD23 shedding was impaired by a broad-spectrum metalloprotease inhibitor, BB-94, by 63% and the ADAM10 inhibitor, GI254023X, by 67%. RT-PCR confirmed the expression of ADAM10 in B cells. P2X7-induced CD23 shedding was not dependent on changes in extracellular Na+, K+ and Ca2+ concentrations, and was unaffected by a panel of enzyme inhibitors targeting various kinases and phospholipases previously implicated in P2X7-mediated signaling events. ATP also induced the rapid shedding of the ADAM10 substrate, CXCL16, with a t1/2 of 1 min. AZ10606120 impaired ATP-induced CXCL16 shedding by 86%. Moreover, P2X7-induced CXCL16 shedding was impaired by BB-94 by 77% and GI254023X by 87%. This data indicates that human P2X7 activation induces the rapid shedding of CD23 and CXCL16, and that this process involves ADAM10. Thus, P2X7 may represent a novel target in disorders involving CD23 or CXCL16

‘Off-the-Shelf’ Immunotherapy: Manufacture of CD8+ T Cells Derived from Hematopoietic Stem Cells

Cellular immunotherapy is revolutionizing cancer treatment. However, autologous transplants are complex, costly, and limited by the number and quality of T cells that can be isolated from and expanded for re-infusion into each patient. This paper demonstrates a stromal support cell-free in vitro method for the differentiation of T cells from umbilical cord blood hematopoietic stem cells (HSCs). For each single HSC cell input, approximately 5 × 104 T cells were created with an initial five days of HSC expansion and subsequent T cell differentiation over 49 days. When the induced in vitro differentiated T cells were activated by cytokines and anti-CD3/CD28 beads, CD8+ T cell receptor (TCR) γδ+ T cells were preferentially generated and elicited cytotoxic function against ovarian cancer cells in vitro. This process of inducing de novo functional T cells offers a possible strategy to increase T cell yields, simplify manufacturing, and reduce costs with application potential for conversion into chimeric antigen receptor (CAR)-T cells for cancer immunotherapy and for allogeneic transplantation to restore immune competence

Group A streptococcus modulates host inflammation by manipulating polymorphonuclear leukocyte cell death responses

Polymorphonuclear leukocyte (PMN) cell death strongly influences the resolution of inflammatory episodes, and may exacerbate adverse pathologies in response to infection. We investigated PMN cell death mechanisms following infection by virulent group A Streptococcus (GAS). Human PMNs were infected in vitro with a clinical, virulent GAS isolate and an avirulent derivative strain, and compared for phagocytosis, the production of reactive oxygen species (ROS), mitochondrial membrane depolarization and apoptotic markers. C57BL/6J mice were then infected, in order to observe the effects on murine PMNs in vivo. Human PMNs phagocytosed virulent GAS less efficiently, produced less ROS and underwent reduced mitochondrial membrane depolarization compared with phagocytosis of avirulent GAS. Morphological and biochemical analyses revealed that PMNs infected with avirulent GAS exhibited nuclear fragmentation and caspase-3 activation consistent with an anti-inflammatory apoptotic phenotype. Conversely, virulent GAS induced PMN vacuolization and plasma membrane permeabilization, leading to a necrotic form of cell death. Infection of the mice with virulent GAS engendered significantly higher systemic pro-inflammatory cytokine release and localized infiltration of murine PMNs, with cells associated with virulent GAS infection exhibiting reduced apoptotic potential. Avirulent GAS infection was associated with lower levels of proinflammatory cytokines and tissue PMN apoptosis. We propose that the differences in PMN cell death mechanisms influence the inflammatory responses to infection by GAS

Roles of extracellular nucleotides and P2 receptors in ectodomain shedding

Ectodomain shedding of integral membrane receptors results in the release of soluble molecules and modification of the transmembrane portions to mediate or modulate extracellular and intracellular signalling. Ectodomain shedding is stimulated by a variety of mechanisms, including the activation of P2 receptors by extracellular nucleotides. This review describes in detail the roles of extracellular nucleotides and P2 receptors in the shedding of various cell surface molecules, including amyloid precursor protein, CD23, CD62L, and members of the epidermal growth factor, immunoglobulin and tumour necrosis factor families. This review discusses the mechanisms involved in P2 receptor-mediated shedding, demonstrating central roles for the P2 receptors, P2X7 and P2Y2, and the sheddases, ADAM10 and ADAM17, in this process in a number of cell types