GI254023X

Activation of the P2X7 receptor induces the rapid shedding of CD 23 from human and murine B cells

CD23 is a ‘low affinity’, transmembrane receptor for IgE that is expressed on B cells and other leukocytes.1 Transmembrane CD23 can be released from the cell surface to form soluble CD23, which also binds IgE, and exerts cytokine-like activities on B cells and other leukocytes.1 Soluble CD23 sustains growth of B-cell precursors,2 promotes B and T cell differentiation,3,4 and drives cytokine release from monocytes.5 The release of CD23 is mediated by membrane metalloproteases of the ADAM (a disintegrin and metalloprotease) family. ADAM 10 is principally responsible for the constitutive and calcium-induced shedding of CD23.

Damage-associated molecular patterns are essential in inflammation and immunity, and function as signals for cell stress, tissue injury and disease.8 Extracellular adenosine 5′-triphosphate (ATP) is a well- characterised damage-associated molecular pattern that activates the P2X7 receptor, a trimeric cation channel, which plays important roles in health and disease.9 Activation of P2X7 by extracellular ATP causes the uptake of organic cations such as ethidium4 and YO-PRO-124.10 P2X7 activation induces a number of downstream effects including the shedding of CD23 from human malignant B cells11’12 and human dendritic cells.13,14 Recently, our group demonstrated that P2X7- induced CD23 shedding from the human multiple myeloma cell line, RPMI 8226, is mediated by ADAM10.15 2′ (3′)-0-(4-Benzoylbenzoyl) ATP (BzATP)-induced CD23 shedding from murine B cells is also mediated by ADAM 10,16,17 but a direct role for P2X7 in this process was not established in these studies. Therefore, it remains unknown if P2X7 activation induces CD23 shedding from murine B cells, as well as from human B cells. The current study demonstrates that P2X7 activation induces the rapid shedding of CD23 from primary human and murine B cells and that this process possibly involves ADAM 10.

R E S U L T S

P2X7 activation induces rapid CD23 shedding from human B cells ATP and BzATP at concentrations of 1 m M and 0.3 m M , respectively, cause near-maximal activation of human P2X7.18 Therefore, to first determine if ATP induces CD23 loss from human B cells, peripheral blood mononuclear cells (PBMCs) were incubated with 1 m M ATP for up to 30 min and cell surface expression assessed by flow cytometry (Figure la). ATP induced a rapid loss of cell surface CD23 from B cells with a t\/2 of approximately 6 min (Figure lb).

To determine whether ATP-induced CD23 loss was mediated by P2X7, PBMCs were incubated for 6 min (the ti/2) in the absence or presence of 1 m M ATP, the most potent P2X7 agonist 0.3 m M BzATP or the non-P2X7 agonists 1 m M adenosine diphosphate (ADP) and uridine 5′-triphosphate (UTP). ATP and BzATP induced a 49 + 9% and 60 ± 9% loss of cell surface CD23, respectively, whereas ADP and UTP had no effect on CD23 expression compared with cells incubated without nucleotide (Figure lc).

Figure 1 P2X7 activation induces CD23 shedding from human B cells, (a, b) PBMCs in NaCI medium were incubated for up to 30 min at 37 °C in the absence or presence of 1 mM ATP as indicated, (c) Cells in NaCI medium were incubated in the absence (basal) or presence of 1 it im ATP, 0.3 mM BzATP, Im M ADP or 1 mM UTP at 37 °C for 6 min. (d) PBMCs in NaCI medium were pre-incubated at 37 °C for 15 min in the absence or presence of 100 nM A Z 10606120 (AZ) and then in the absence (basal) or presence of 1 mM ATP for 6 min at 37 °C. (a-d) Incubations were stopped by addition of MgCI2 medium and centrifugation. PBMCs were labelled with APC-conjugated anti-CD19, phycoerythrin-conjugated anti-CD23 or isotype control mAb and 7AAD. The mean fluorescence intensity of cell surface CD23 expression on CD19+7AAD” B cells was determined by flow cytometry, (e) PBMCs in NaCI medium were incubated in the absence (basal) or presence of 1 mM ATP at 37 °C for 20 min, incubations were stopped by centrifugation and the amount of soluble CD23 in cell-free supernatants was determined by ELISA, (a) Histograms (from one representative individual) show CD23 expression on (black or grey fill) or isotype control mAb binding (black line) to CD19+7AAD” PBMCs incubated in the absence (black fill or black line) or presence (grey fill) of 1 mM ATP for 30 min; isotype control mAb binding to CD19+7AAD^ PBMCs incubated in the presence of ATP was similar to that of cells incubated in the absence of ATP (not shown), (b-d) M ean±s.d. (three individuals) or (e) three individuals; *P< 0 .05 and **P < 0 .01 compared with basal, and f P < 0 .0 5 compared with ATP alone. The P2X7 antagonist AZ10606120,19 at a concentration of 100 nM results in near-complete inhibition of human P2X7.20 Therefore, to confirm that ATP-induced loss of CD23 was mediated by P2X7, PBMCs were pre-incubated in the absence or presence of 100 nM AZ10606120, and 1 mM ATP-induced CD23 loss determined by flow cytometry. AZ10606120 impaired ATP-induced CD23 loss by 89+12% (Figure Id). In the absence of ATP, AZ10606120 did not alter CD23 expression (Figure Id). To determine if the P2X7-induced loss of cell surface CD23 was due to CD23 shedding, PBMCs were incubated in the absence or presence of 1 mM ATP for 20 min, and the relative amount of soluble CD23 in cell-free supernatants quantified by enzyme-linked immunosorbent assay (ELISA). Incubation of cells with ATP resulted in a significandy higher release of soluble CD23 compared with cells incubated in the absence of ATP (Figure le). Murine B cells express functional P2X7 established,21 but less well known for murine B cells. Similar to human P2X7, ATP and BzATP at concentrations of 1 m M and 0.3 m M, respectively, result in near-maximal activation of murine P2X7,18 whereas the P2X7 antagonist AZ10606120, at a con­ centration of 10 pM , results in near-complete inhibition of murine P2X7.22 Therefore, to first test whether functional P2X7 was present on murine B cells from C57BL/6 and DBA/1 mice, splenic cells from these two strains were pre-incubated in the absence or presence of 10 pM AZ10606120, and 1 mM ATP-induced YO-PRO-l2+ uptake into B cells was determined by flow cytometry. C57BL/6 mice were studied because of the availability of the Pfizer P2X7 knockout mice,23 which had been backcrossed onto a C57BL/6 background.24 DBA/1 mice were also studied as this strain has been used to demonstrate a role for CD23 in rheumatoid arthritis,25 a disease in which P2X7 is also thought to be involved.26,27 ATP induced significant YO-PRO-l2+ uptake into murine B cells, from either C57BL/6 or DBA/1 mice, compared with YO-PRO-l2+ uptake in the absence of ATP (Figures 2a and b). Moreover, 10 pM AZ10606120 significantly impaired ATP-induced YO-PRO-l2+ uptake in B cells from C57BL/6 or DBA/1 mice, by 88 ± 14% or 95 ±8%, respectively (Figures 2a and b). In the absence of ATP, AZ10606120 did not alter YO-PRO-l2+ uptake in B cells from either mouse strain (Figures 2a and b). P2X7 activation induces rapid CD23 shedding from murine B cells To determine if ATP induces CD23 loss from murine B cells, splenic cells were incubated with 1 m M ATP for up to 30 min and cell surface CD23 expression was assessed by flow cytometry (Figures 3a and b). ATP induced a rapid loss of cell surface CD23 from B cells from both C57BL/6 and DBA/1 mice with a ti/2 of approximately 7 min (Figure 3c). To determine whether ATP-induced CD23 loss was mediated by P2X7, cells from both mouse strains were incubated for 7 min (the fi/2) in the absence or presence of 1 h im ATP, 0.3 m M BzATP, 1 m M ADP or 1 mM UTP. Similar to above (Figure 3c), ATP induced a 37 ± 4% and 57 ± 4% loss of cell surface CD23 from B cells from C57BL/6 and DBA/1 mice, respectively (Figures 3d and e). BzATP induced a 33 ± 8% and 58 ± 5% loss of cell surface CD23 from B cells from C57BL/6 and DBA/1 mice, respectively, whereas ADP and UTP had no effect compared with cells incubated without nucleotide in either mouse strain (Figures 3d and e). To confirm that ATP-induced loss of CD23 was mediated by P2X7, cells were pre-incubated in the absence or presence of 10 p M AZ10606120, and 1 mM ATP-induced CD23 loss was determined by flow cytometry. AZ10606120 impaired ATP-induced CD23 loss by 95 ± 8 % and 97 ± 6% in B cells from C57BL/6 and DBA/1 mice, respectively (Figures 3f and g). In the absence of ATP, AZ10606120 did not alter CD23 expression in B cells from either mouse strain (Figures 3f and g). Finally, to further confirm a role for P2X7 in ATP-induced CD23 loss, P2X7 knockout mice were used. Flow cytometric measurements demonstrated that the amount of CD23 expression between control- treated B cells from wild-type and P2X7 knockout mice was similar (Figure 3h). In contrast, incubation with 1 m M ATP induced a loss of cell surface CD23 in B cells from wild-type mice (34 ± 10% loss) but ATP-induced CD23 loss from B cells from P2X7 knockout mice was minimal (8 + 7% loss) (Figure 3h). Figure 2 C57BL/6 and DBA/1 murine B cells express functional P2X7. (a, b) Splenic cells in NaCI medium were pre-incubated in the absence or presence of 10 h m A Z 10606120 (AZ) at 37 °C for 15 min. Cells were then incubated with 1 (j m Y0-PR 0-12+ in the absence (basal) or presence of 1 mM ATP at 37 °C for 15 min. Incubations were stopped by the addition of MgCI2 medium and centrifugation. Cells were labelled with APC-conjugated anti- CD19 mAb and 7AAD, and the mean fluorescence intensity of Y0-PR 0-1Z+ uptake into CD19+7AAD_ B cells was determined by flow cytometry. Results are mean + s.d. ((a) three mice or (b) duplicate values from two mice); " P < 0 . 0 1 compared with control, and n P < 0 .0 1 compared with ATP alone. To indirectly assess if the ATP-induced loss of CD23 from murine B cells was a result of CD23 shedding or internalisation, splenic cells from C57BL/6 mice were incubated in the absence or presence of 1 mM ATP for 20 min and the expression of cell surface CD23 on fixed cells or total CD23 in fixed and permeabilised cells was examined by flow cytometry. CD23 expression was similar in fixed cells compared with fixed and permeabilised cells (Figure 4a) suggesting that the P2X7-induced loss of CD23 was due to CD23 shedding rather than internalisation. To directly determine if the P2X7-induced loss of cell surface CD23 was due to CD23 shedding, splenic cells from C57BL/6 wild- type and P2X7 knockout mice were incubated in the absence or presence of 1 m M ATP for 20 min, and the relative amount of soluble CD23 in cell-free supernatants was quantified by ELISA. Incubation of wild-type cells with ATP resulted in a significantly higher release of soluble CD23 compared with P2X7 knockout cells incubated with ATP, or cells of either strain incubated in the absence of ATP (Figure 4b). Moreover, the amount of soluble CD23 release from P2X7 knockout cells incubated in the absence or presence of ATP was similar to that of wild-type cells incubated in the absence of ATP (Figure 4b). Splenic cells from P2X7 knockout mice do not express full-length, functional P2X7 Recent evidence indicates that Pfizer P2X7 knockout mice,23 from which the P2X7 knockout mice in the current study were derived,24 express three novel C-terminal-truncated P2X7 variants termed 13A (—60 kDa), 13B (~60 kDa) and hybrid (~70 kDa) P2 x 7.28 Pfizer P2X7 knockout mice were generated by targeting exon 13 of the P2RX7 gene,23 and polymerase chain reaction (PCR) with primers to exons 9 and 11 of the P2RX7 gene demonstrates P2X7 expression in various tissues from these mice.28 Consistent with these findings, quantitative real-time (RT)-PCR with primers to exons 1 and 2 of the P2RX7 gene revealed significant expression of P2RX7 transcripts in the splenic cells from both wild-type and P2X7 knockout mice (Figure 5a). Relative P2X7 expression was twofold higher in P2X7 knockout splenic cells compared with wild-type splenic cells, but this failed to reach statistical significance (Figure 5a). These data indicate the potential presence of escape variants of P2X7 in the P2X7 knockout mice used to study ATP-induced CD23 shedding above. To determine if the mRNA transcripts for P2X7 detected above (Figure 5a) were translated into protein, splenic cells of these mice were examined by immunoblotting with an antibody against the extracellular domain of murine P2X7, which can detect full-length P2X7, as well as the C-terminal-truncated P2X7 variants.28 Murine RAW 264.7 macrophages, which are known to express full-length P2X7,29 were included as a positive control. Immunoblotting identified a major band at 81 kDa, corresponding to glycosylated full-length P2X7, in wild-type splenic cells and RAW 264.7 macrophages, but not in P2X7 knockout splenic cells (Figure 5b). Immunoblotting also revealed a major band at 75 kDa in all three cell types (Figure 5b). The size of this band corresponds to that of the hybrid C-terminal-truncated P2X7 variant present in Pfizer P2X7 knockout mice. However, this variant contains a short sequence of the targeting vector originally used to disrupt the P2RX7 gene;28 thus, this variant cannot be present in wild-type splenic cells or RAW 264.7 macrophages. Therefore, this 75-kDa band most likely represents non-specific binding. Immunoblotting also detected a major band at 154 kDa in wild-type and P2X7 knockout splenic cells, but not in RAW264.7 macrophages (Figure 5b). This band does not correspond to any known P2X7 variant and thus most likely represents non-specific binding. Flow cytometric measurements of ATP-induced YO-PRO-l2+ uptake were then used to confirm the absence of functional P2X7 in splenic B cells from the P2X7 knockout mice used to study ATP- induced CD23 shedding above. T cells from GlaxoSmithKline P2X7 knockout mice, derived by targeting exon 1 of the P2RX7 gene,30 express P2X7k, an alternate splice variant that has escaped gene inactivation and encodes functional receptors.31-33 Therefore, ATP- induced YO-PRO-l2+ uptake into splenic T cells was also assessed. As expected, I m M ATP induced YO-PRO-l2+ uptake into both B and T cells from wild-type mice (Figure 5c). In contrast, 1 mM ATP failed to induce YO-PRO-l2+ uptake into either B or T cells from P2X7 knockout mice (Figure 5c). Collectively, these results indicate that the P2X7 knockout mice used to study ATP-induced CD23 shedding above do not express full-length, functional P2X7. ADAM10 may mediate P2X7-induced CD23 shedding from human and murine B cells To determine a role for metalloproteases in P2X7-induced CD23 shedding, human PBMCs or murine splenic cells were pre-incubated in the absence or presence of the broad-spectrum metalloprotease antagonist, BB-94,34 or the ADAM10 antagonist, GI254023X,35 and 1 mM ATP-induced CD23 shedding from B cells was determined by flow cytometry. BB-94 (1 pM) impaired P2X7-induced CD23 shedding from B cells by 66 ± 10, 100 ± 0 and 96 ± 6% from human, C57BL/6 and DBA/1 mice, respectively (Figures 6a-c). GI254023X (3 pm ) impaired P2X7-induced CD23 shedding from B cells by 77 ± 20, 83 ± 29 and 100 ± 0 % from human, C57BL/6 and DBA/1 mice, respectively (Figures 6d—f). In the absence of ATP, BB-94 did not alter basal CD23 expression in human and murine cells (Figures 6a-c). Likewise, GI254023X did not alter basal CD23 expression in human or DBA/1 B cells (Figures 6d and f). In contrast, GI254023X significantly reduced basal (constitutive) CD23 shedding from C57BL/6 B cells (Figure 6e). Figure 5 Splenic cells from P2X7 knockout mice do not express full-length, functional P2X7. (a) Relative P2X7 expression in the spleens of wild-type (WT) and P2X7 knockout (KO) C57BL/6 mice was measured by quantitative RT-PCR using primers to exons 1 and 2 of the P2RX7 gene, (b) Whole lysates of WT and P2X7 KO splenic cells, and RAW 264.7 macrophages (RAW) were examined by immunoblotting using an antibody agonist the extracellular epitope of P2X7. (c) Splenic cells, from WT and P2X7 KO mice, in NaCI medium were incubated with 1 pMYO-PRO-lz+ in the absence or presence of 1 itim ATP at 37 °C for 15 min. Incubations were stopped by the addition of MgCl2 medium and centrifugation. Cells were labelled with peridinin chlorophyll protein/ cyanine 5.5-conjugated anti-CD3 mAb and APC-conjugated anti-CD19 mAb, and the mean fluorescence intensity of Y0-PR0-12+ uptake into CD3“ CD19+ B cells and CD3+CD19~ T cells was determined by flow cytometry. Results are (a, c) mean±s.d. (three mice; points represent individual mice) or (b) one representative of two experiments. Figure 6 Metalloproteases mediate P2X7-induced shedding from human and murine B cells, (a, d) Human PBMCs, and (b, e) C57BL76 or (c, f) DBA/1 murine splenic cells were pre-incubated in the presence of (a-f) dimethyl sulphoxide (DMSO), (a-c) 1 pM BB-94 (BB) or (d-f) 3pM GI254023X (Gl), and (a-f) then in the absence (basal) or presence of 1 it im ATP at 37 °C for (a, d) 6 or (b, c, e, f) 7 min. Incubations were stopped by addition of MgCl2 medium and centrifugation. B cells were labelled with APC-conjugated anti-CD19 mAb, phycoerythrin- or fluorescein isothiocyanate-conjugated anti-CD23 or isotype control mAb and 7AAD. The mean fluorescence intensity of cell surface CD23 expression on CD19+7AAD“ B cells was determined by flow cytometry. Results are mean±s.d. ((a) four or (d) three individuals, or (b, c, e, f) three mice); *P < 0 .05 and **P < 0 .01 compared with corresponding basal, and n P<0.01 compared with ATP in the presence of DMSO. DISCUSSION The current study shows for the first time that P2X7 activation induces the rapid shedding of CD23 from primary human and murine B cells. This was confirmed by a series of experiments. First, ATP induced the rapid cell surface loss of CD23 shedding with a f1/2 of approximately 6 and 7 min from human and murine B cells, respectively. Second, the most potent P2X7 agonist BzATP also induced the rapid cell surface loss of CD23, whereas the non-agonists ADP and UTP had no effect Third, a specific P2X7 antagonist, AZ10606120,19 almost completely impaired ATP-induced CD23 shedding in both human and murine B cells. Fourth, ATP failed to induce CD23 shedding from B cells from C57BL/6 P2X7 knockout mice. Finally, measurements of soluble CD23 (as well as total CD23 expression for murine B cells) indicated that cell surface loss of CD23 was due to shedding from both human and murine B cells. Using the ADAM 10 inhibitor, GI254023X,35 the current study also shows that P2X7-induced CD23 shedding from human and murine B cells is possibly mediated, at least in part, by ADAM 10. This finding is consistent with a role for ADAM10 in P2X7-induced CD23 shedding from human RPMI 8226 myeloma cells.15 Remarkably, the rate of P2X7-induced CD23 shedding from human and murine B cells was similar to that observed for RPMI 8226 cells (fi/2 of ~ 7 min) indirectly supporting the involvement of a common mechanism. A potential role for ADAM10 in P2X7-induced CD23 shedding from primary B cells is indirectly supported by other studies. ATP-induced CD23 shedding from human leukaemic monocytic U937 cells7 and BzATP-induced CD23 shedding from Chinese hamster ovary cells16 and murine B cells17 are also mediated by ADAM 10. The possibility remains that other metalloproteases may also be involved in P2X7-induced CD23 shedding from human and murine B cells. ADAM 8, 15, 28 and 33 have been associated with constitutive CD23 shedding from CD23-transfected HEK293 cells36 and primary murine embryonic fibroblasts,6 although ADAM 10 was identified as the principal sheddase of CD23. It remains unknown how P2X7 potentially activates ADAM 10. Repeated attempts using a Fluorogenic Peptide Substrate III assay (R&D Systems, Minneapolis, MN, USA) (which detects the activity of ADAM10, as well as ADAM8, 9 and 17) were unable to demonstrate that ATP could stimulate ADAM 10 activation on RPMI 8226 cells despite detectable ADAM activity on these cells in the absence of ATP (Pupovac and Sluyter, unpublished observations). Thus, the possibility remains that P2X7 activation induces cell surface CD23 shedding through the colocalisation of ADAM10 and CD23, rather than by directly stimulating ADAM 10 activity. Finally, using a candidate approach, our previous studies also failed to identify potential intracellular signalling molecules involved in P2X7-induced CD23 shedding.20 The current study demonstrates the presence of functional P2X7 on murine B cells from both C57BL/6 and DBA/1 mice. Although the presence of functional P2X7 on human B cells is well established, reports of functional P2X7 on murine B cells are limited. In the current study, P2X7 activation induced CD23 shedding from murine B cells. Moreover, ATP was shown to induce YO-PRO-I21' uptake into murine B cells, and that the process was impaired by AZ10606120 and in B cells from P2X7 knockout mice. Of note, the relative amounts of P2X7-mediated YO-PRO-l2+ uptake and rates of P2X7-mediated CD23 shedding were similar between C57BL/6 and DBA/1 mice. This similar amount of relative P2X7 function most likely reflects the presence of the partial loss-of-function mutation P451L in both of these strains. A previous study has shown that lipopolysaccharide can induce CD23 shedding from human and murine B cells, and that this process is mediated by Toll-like receptor 4 (TLR4) and matrix metalloprotease 9.38 However, it remains unlikely that lipopolysaccharide, potentially present in the reagents used, was responsible for the ATP-induced CD23 observed in the current study. First, ATP induced the rapid (< 30 min) shedding of CD23; in contrast, lipopolysaccharide induces the slow (24 h) shedding of CD23.38 Second, ADP and UTP failed to induce CD23 shedding, whereas ATP-induced shedding was impaired by P2X7 antagonists and in B cells from P2X7 knockout mice. Nevertheless, future studies using B cells from either C3H/HeJ mice, which are hyporesponsive to lipopolysaccharide because of a single point mutation in the TLR4 gene,39 or TLR4 knockout mice will be of value to address this potential issue further. Human genetic27,40 and murine model studies26,41 suggest a role for P2X7 in rheumatoid arthritis and Sjogren’s syndrome. The role of P2X7 in these disorders has largely been attributed to the release of the proinflammatory cytokines, interleukin-1|3 and interleukin-18.26,42 However, synovial or circulating soluble CD23 is elevated in patients with rheumatoid arthritis43,44 or Sjogren’s syndrome.45 Moreover, B cells also play major roles in the pathogenesis of these disorders through the production of autoantibodies.46,47 Thus, the possibility remains that P2X7-induced shedding of pro-inflammatory soluble CD23 from B cells may also be involved in rheumatoid arthritis, Sjogren’s syndrome or other disorders. However, evidence directly linking B cells, P2X7 and CD23 in inflammatory and autoimmune disease is lacking. An alternate, but not mutually exclusive possibility is that ATP-induced CD23 shedding is an early event of P2X7-mediated apoptosis. In the current study, 30 min incubation with ATP caused a small but significant amount of apoptosis, determined by forward scatter (cell shrinkage) and 7-aminoactinomycin D (7AAD) uptake (loss of membrane integrity),48 in murine but not human B cells compared with respective B cells incubated in the absence of ATP (results not shown). In contrast, human and murine B-cell apoptosis was not increased following 6-7 min treatment with either ATP or BzATP compared with control treatment, nor was B-cell apoptosis increased following incubation with AZ10606120 in the absence or presence of ATP (results not shown). Nevertheless, given that P2X7 activation can induce apoptosis in human PBMCs49 and murine splenic cells,50 the possibility remains that ATP-induced CD23 shed­ ding is an early upstream event in P2X7-mediated apoptosis of B cells. In conclusion, this study demonstrates human and murine P2X7 activation induces the rapid shedding of CD23 from B cells. Moreover, the study indicates a potential role for ADAM10 in this process. METHODS Reagents Ficoll Paque™ PLUS was from GE Healthcare Bio-Sciences (Uppsala, Sweden). DMEM/F12 medium, YO-PRO-1 iodide, TRIzol reagent and TaqMan Uni­ versal Master Mix II with uradl-DNA glycosylase and Gene Expression Assays were from Life Technologies (Grand Island, NY, USA). Red blood cell lysis buffer, ATP, BzATP, ADP, UTP, paraformaldehyde and RNALifer were from Sigma-Aldrich (St. Louis, MO, USA). Dimethyl sulphoxide and Tween-20 were from Amresco (Solon, OH, USA). AZ10606120 and BB-94 (Batimastat) were from Tocris Bioscience (Ellisville, MO, USA). GI254023X was kindly provided by GlaxoSmithKline (Stevenage, UK). 7AAD was from Enzo Life Sciences (Plymouth Meeting, PA, USA). Phycoerythrin-conjugated murine anti-human CD23 (clone EBVCS2), phycoerythrin-conjugated isotype control (clone P3.6.2.8.1), fluorescein isothiocyanate-conjugated rat anti-murine CD23 (clone B3B4), fluorescein isothiocyanate-conjugated isotype control (clone eBR2a), allophycocyanin (APC)-conjugated m urine anti-human CD19 (clone HIB19) and APC-conjugated rat anti-murine CD19 (clone eBiolD3) monoclonal antibodies (mAb) were from eBioscience (San Diego, CA, USA). Peridinin chlorophyll protein/cyanine 5.5-conjugated hamster anti-murine CD3 (clone 145 -201 ) mAb was from BioLegend (San Diego, CA, USA). Rabbit anti­ murine P2X7 polyclonal antibody (extracellular domain) was from Alomone Labs (Jerusalem, Israel).

Cells

All experiments involving humans and mice were approved by the University of Wollongong ethics committee. Human peripheral blood was collected into VACUETTE* lithium heparin tubes (Greiner Bio-One, Frickenhausen, Germany) and diluted with an equal volume of phosphate-buffered saline (PBS). PBMCs were separated by density gradient centrifugation over Ficoll-Paque™ PLUS (560 x g for 30 min), washed once in PBS (450 x g for 10 min) and then twice in NaCl medium (145 m M NaCl, 5 m M KC1, 5 m glucose, 10 n u HEPES, pH 7.4). The mean percentage of CD19+ cells within the 13 PBMC preparations used was 4.0% (range 2.5—7.5%). C57BL/6 and DBA/1 mice were from Animal Resources Centre (Perth, WA, Australia) or Australian BioResources (Moss Vale, NSW, Australia). P2X7 knockout mice23 backcrossed onto a C57BL/6 background, were bred at the Centenary Institute (Sydney, NSW, Australia) or the University of Wollongong (Wollongong, NSW, Australia).24 Mice were killed and spleens collected in ice-cold PBS. Spleens were teased apart using a needle and forceps in ice-cold PBS, and cells were filtered through a 70-pm nylon cell strainer (BD, San Jose, CA, USA). Splenic cells were then washed in ice-cold PBS (400 xg for 5 min) and the pellet resuspended in red blood cell lysis buffer. Red blood cells were lysed for 3 min at room temperature with agitation and the remaining leukocytes were washed once with ice-cold DMEM/F12 medium. Cells were then washed twice in NaCl medium (for functional assays) or PBS (for PCR or immunoblotting). Murine RAW 264.7 macrophages (American Type Culture Collection, Manassas, VA, USA) were maintained as described.24

Measurement of nucleotide-induced CD23 shedding by flow cytometry

Nucleotide-induced CD23 shedding from cells was indirectly assessed by flow cytometric measurements of ATP-induced loss of cell surface CD23 as described.1 Briefly, PBMCs or splenic cells suspended in NaCl medium (1 X 106 cells per ml), were incubated in the absence or presence of nucleotide (as indicated) for up to 30 min at 37 °C. In some experiments, cells in NaCl medium were pre-incubated at 37°C for 15 min in the absence or presence of antagonist, and then in the absence or presence of 1 m M ATP (as indicated). Incubations with nucleotide were stopped by addition of an equal volume of ice-cold MgCL medium (NaCl medium containing 20 m M MgCL) and centrifugation. Cells were then washed once with NaCl medium and incubated with species-specific fluorochrome-conjugated anti-CD23 or isotype control mAb, anti-CD 19 mAb and 7AAD for 30 min at 4°C. The mean fluorescence intensity of cell surface CD23 expression on viable CD19+7AAD_ cells was determined using a BD LSRII flow cytometer (using band-pass filters 575/26 or 515/20 nm for CD23, 660/20 nm for CD19 and 695/40 nm for 7AAD) and Flowjo software (Tree Star, Ashland, OR, USA).

Cell surface and total CD23 expression following treatment were assessed as previously described.51 Splenic cells were washed once with NaCl medium and labelled with APC-conjugated anti-murine CD19 and 7AAD for 30 min at 4°C. Cells were fixed by suspension in ice-cold 0.25% paraformaldehyde in PBS for 1 h at 4 °C. Cells were then either washed once in cold PBS (fixed) or in 0.2% Tween-20 in PBS (fixed and permeabilised) and labelled with fluorescein isothiocyanate-conjugated anti-murine CD23 or isotype control mAb for 30 min at 4°C. The mean fluorescence intensity of cell surface and total (cell surface and intracellular) CD23 expression on CD19+ cells was determined using flow cytometry (using band-pass filters 515/20 nm for CD23, 660/20 nm for CD 19 and 695/40 nm for 7AAD) and Flowjo software.

Measurement of soluble CD23 by ELISA

PBMCs or splenic cells suspended in NaCl medium (5 x 106 cells per ml) were incubated in the absence or presence of 1 m M ATP for 20 min at 37 °C. Incubations were stopped by centrifugation ( l l OOOxg for 10s). Cell-free supernatants were stored at – 80°C until required. Soluble CD23 was quantified using the Hum an CD23/Fce RII Quantikine ELISA Kit or the Mouse CD23/Fce RII DuoSet ELISA Development Kit (both R&D Systems), according to the manufacturer’s instructions.

Measurement of P2X7 pore formation by flow cytometry

P2X7-induced pore formation in murine B cells was assessed by flow cytometric measurements of ATP-induced YO-PRO-l24 uptake as described.52 Briefly, splenic cells suspended in NaCl medium (1 x 106 cells per ml) were pre- incubated in the absence or presence of 10 pM AZ10606120 (as indicated), and then with 1 pM YO-PRO-124 in the absence or presence of ATP (as indicated) for 15 min at 37 °C. Incubations were stopped by addition of an equal volume of ice- cold MgCl2 medium and centrifugation. Cells were washed once with NaCl medium. Cells were labelled with APC-conjugated anti-murine CD19 mAb (and 7AAD or in some experiments, with peridinin chlorophyll protein/cyanine 5.5- conjugated anti-murine CD3 mAb) and washed once with NaCl medium. The mean fluorescence intensity of YO-PRO-124 uptake into CD19+ cells (or in some experiments, CD19+CD3~ or CD19”CD3+ cells) was determined using flow cytometry (using band-pass filters 515/20 nm for YO-PRO-12+, 695/40 nm for CD3 and 660/20 nm for CD19) and Flowjo software.

Measurement of P2X7 by quantitative RT-PCR

Mouse spleens were stored in RNAlufer at —20°C until required. Total RNA was isolated using TRIzol reagent according to the manufacturer’s instructions. cDNA synthesis was performed using a qScript cDNA Synthesis Kit (Quanta Biosciences, Gaithersburg, MD, USA) following the manufacturer’s instruc­ tions. RT-PCR reactions consisted of 5 pi 2 x TaqMan Universal Master Mix II with uracil-DNA glycosylase, 0.5 pi of 20 x TaqMan Gene Expression Assay (specific primers/probes), 2 pi cDNA and sterile water in a total volume of 10 pi. Each PCR reaction was performed in triplicate. Quantitative RT-PCR was performed using standard primers for murine P2RX7 (FAM-labelled; Mm 00440578_ml) and murine GAPDH (VIC-labelled; Mm99999915_gl). RT-PCR was performed on an Eco Real-time PCR System (Illumina, San Diego, CA, USA) in sealed 48-well plates (Gene Target Solutions, Dural, NSW, Australia). The PCR cycle consisted of an initial uracil-DNA glycosylase incubation step of 50 °C for 2 min, polymerase activation step of 95 °C for 10 min and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Relative gene expression was normalised to the murine GAPDH housekeeping gene and determined using Eco Software v5.0 (Illumina).

Measurement of P2X7 by immunoblotting

Immunoblotting of murine cells was performed as described.53 Briefly, whole cell lysates were separated under reducing conditions using Any kD Mini- PROTEAN TGX Stain-Free Gels (Bio-Rad, Hercules, CA, USA) and transferred to nitrocellulose membrane (Bio-Rad), and immunoblotting was performed using an anti-P2X7 antibody. To confirm equal protein loading between splenic cell samples within stain-free gels, total protein was assessed using a Bio-Rad Criterion Stain Free Imager (results not shown).

Presentation of data and statistics

Data are presented as mean ± s.d. from individual people or mice except where stated. Differences between groups were compared using an unpaired Student’s t-test for single comparisons or one-way analysis of variance (using Tukey’s multiple comparison test) except for human ELISA data, for which a paired Student’s t-test was used. Statistical comparisons were performed using Prism 5 (Windows version 5.01; GraphPad Software, San Diego, CA, USA) with P < 0 .05 considered significant.