Human P2X7 receptor activation induces the rapid shedding of CXCL16

Abstract

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.

CAY10593 inhibits the human P2X7 receptor independently of phospholipase D1 stimulation

“…ATP-induced CD23 shedding was indirectly assessed by measuring the loss of cell-surface CD23 using an anti-CD23 mAb and flow cytometry following 7 min incubation with 1 mM ATP, which is approximate to the t 1/2 for this process [17,29]. As previously observed [17,29], ATP induced CD23 shedding from RPMI 8226 cells in NaCl medium (Fig.…”

“…More recently, we have shown that P2X7-induced CD23 shedding is mediated by ADAM10 [29]. However, it is unknown if P2X7-induced CD23 shedding results from changes in intracellular cation concentrations, stimulation of signalling pathways downstream of P2X7 activation or by a direct physical interaction of P2X7 itself.…”

“…We have previously shown that human RPMI 8226 B cells express P2X7, and that activation of P2X7 on these cells induces pore formation and the shedding of CD23 [17,28,29]. During our preliminary investigations of the possible intracellular signalling enzymes involved in P2X7-induced CD23 shedding from RPMI 8226 cells we observed that the 1-(piperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one analogue and specific PLD1 inhibitor, CAY10593 [30], significantly impaired P2X7-induced CD23 shedding.…”

“…RPMI 8226 cells have been previously shown to express endogenous P2X7 [17,28,29]. Therefore, to test these specific P2X7 antagonists on endogenously expressed P2X7, RPMI 8226 cells were pre-incubated in the absence or presence of increasing concentrations of AZ10606120, AZ11645373 or A-438079, and the ATPinduced ethidium + uptake (pore formation) was measured by flow cytometry.…”

P2X7 receptor activation induces CD62L shedding from human CD4+ and CD8+ T cells

“…Human P2X7 activation induces the rapid shedding of CD23 and CXCL16 from malignant B cells [19][20][21] , as well as the shedding of the interleukin-6 receptor (IL-6R) from human epithelial cells or murine fibroblasts co-transfected with human P2X7 and human IL-6R [22] . Human P2X7-induced CD23, CXCL16 and IL-6R shedding is primarily mediated by ADAM10 [21,22] , however it is likely that ADAM17 is the principle sheddase involved in P2X7-induced CD62L shedding from human CD4 + and CD8 + T cells. It has previously been shown that P2X7-induced CD62L and CD23 shedding from malignant B cells is mediated by different metalloproteases [20] , and that ADAM17 is the principle sheddase for both calcium-and ATP-induced CD62L shedding from murine B cells [23] .…”