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J S Wiley

Publications and source records attributed to J S Wiley.

At least 19 recordsLinked to original sources
PubMed2004

P2X7 receptor polymorphism impairs extracellular adenosine 5'-triphosphate-induced interleukin-18 release from human monocytes.

Interleukin (IL)-18 is an important proinflammatory cytokine processed and released from cells of the monocyte lineage by activation of the P2X(7) receptor by extracellular adenosine 5'-triphosphate (ATP). We examined if a loss-of-function polymorphism of the human P2X(7) receptor (glutamic acid-496 to alanine) impairs this process. Using a whole blood-based assay, ATP-induced release of IL-18 from homozygous subjects after 120 min incubation with ATP was 42% of that from wild-type subjects. Moreover, the level of ATP-induced IL-18 release from lipopolysaccharide (LPS)-primed monocytes of homozygous subjects after 30 and 60 min incubation with ATP was 21 and 44%, respectively, of that from wild-type monocytes. Nigericin, a K(+) ionophore, induced a similar release of IL-18 from monocytes of either genotype. ATP-induced ethidium(+) uptake in LPS-primed, monocytes of homozygous subjects was only 11% of that in wild-type monocytes, while P2X(7) surface expression on LPS-primed, homozygous monocytes was 44% of that on wild-type monocytes.

Adenosine Triphosphate↗
PubMed2003

Specific detection of non-functional human P2X(7) receptors in HEK293 cells and B-lymphocytes.

P2X(7) receptor/channels mediate ATP-induced apoptosis in a range of cells including lymphocytes. HEK293 cells were transfected with wild-type human P2X(7) receptor or site-directed mutant constructs (K193A, K311A and E496A) known to be non-functional from measurements of barium/ethidium influx in the presence of ATP or 2',3'-O-(4-benzoylbenzoyl)-ATP. An antibody was designed against an epitope from a loop adjacent to the extracellular ATP site. The epitope was unavailable in cells expressing normal functional surface receptors. Non-functional surface receptors as well as intracellular receptors selectively bound the antibody. So did B-lymphocytes from chronic lymphocytic leukemia patients expressing non-functional (E496A) mutant receptor.

Adenosine Triphosphate↗
PubMed2003

Autoimmune hemolysis associated with primary biliary cirrhosis responding to ursodeoxycholic acid as sole treatment.

Coombs' positive autoimmune hemolytic anemia (AIHA) has been rarely described in association with primary biliary cirrhosis (PBC). The previously reported cases have responded to treatment with a combination of corticosteroids and ursodeoxycholic acid (UDCA). We report a case of AIHA occurring in association with PBC, which has responded to treatment with UDCA alone. Possible mechanisms of autoimmune hemolysis in this patient include bile salt induced immune dysregulation and direct damage to red cell membranes by bile salts leading to exposure of neoantigens and development of red cell autoantibodies. A trial of UDCA as a single agent should be considered as initial treatment in this rare disorder.

Anemia, Hemolytic, Autoimmune↗
PubMed2002

Point mutations confer loss of ATP-induced human P2X(7) receptor function.

Residues considered essential for ATP binding to the human P2X(7) receptor (hP2X(7)R) were investigated. HEK293 cells or Xenopus oocytes were transfected with wild-type or site-directed mutants of hP2X(7)R constructs and channel/pore activity measured in the presence of ATP or 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP). Barium uptake and ethidium influx into HEK293 cells were abolished in cells expressing K193A and K311A mutants, and were partially reduced in cells expressing mutant P210A. K193A and K311A mutations also completely abolished responses to ATP and BzATP in Xenopus oocytes as measured by electrophysiology. These results indicate that K193 and K311 are essential residues in ATP binding in the hP2X(7)R.

Adenosine Triphosphate↗
PubMed2001

P2Y(11) receptor expression by human lymphocytes: evidence for two cAMP-linked purinoceptors.

The effects of extracellular ATP, ADP, AMP and adenosine on cAMP accumulation have been studied in freshly isolated B-lymphocytes from patients with chronic lymphocytic leukemia. Extracellular ATP and several nucleotide analogs stimulated cAMP accumulation with the following order of potency: ATP (EC(50)=120+/-20 microM)>ADP>>AMP. ADP was less effective than ATP and may be a partial agonist. AMP exhibited variable but generally weak activity. The stable analog of ATP, alpha,beta-methylene ATP (EC(50)=110+/-15 microM) also stimulated cAMP accumulation and exhibited similar efficacy to ATP. The P2Y(2) receptor agonist, UTP had no effect on intracellular cAMP levels. Adenosine and the A(2A)/A(2B) receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA) also stimulated cAMP accumulation in CLL lymphocytes. Adenosine deaminase inhibited the cAMP response to adenosine but had no effect on the ATP-induced cAMP response. On the other hand, the AMP analog, adenosine 5'-thiomonophosphate, (AMPS; 1.0 mM) inhibited ATP-induced and alpha,beta-methylene ATP-induced cAMP production but had no effect on adenosine-induced cAMP production. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the presence of P2Y(11) receptor as well as A(2A) and A(2B) receptor mRNA in chronic lymphocytic leukemia lymphocytes. However, A(2B) receptors would appear to be relatively ineffective because the A(2A) selective agonist, CGS-21680 exhibited comparable efficacy to NECA. Furthermore, the A(2A)-selective antagonist 8-(3-chlorostyryl)-caffeine (CSC) right-shifted the concentration-response curve for NECA. Taken together, the data indicate that ATP induces cAMP accumulation via the activation of P2Y(11) receptors whereas adenosine induces cAMP accumulation via the activation of A(2A) receptors. Coordinate activation of P2Y(11) and A(2A) receptors may influence the developmental fate of normal B-lymphocytes.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2001

A Glu-496 to Ala polymorphism leads to loss of function of the human P2X7 receptor.

The P2X(7) receptor is a ligand-gated cation-selective channel that mediates ATP-induced apoptosis of cells of the immune system. We and others have shown that P2X(7) is nonfunctional both in lymphocytes and monocytes from some subjects. To study a possible genetic basis we sequenced DNA coding for the carboxyl-terminal tail of P2X(7). In 9 of 45 normal subjects a heterozygous nucleotide substitution (1513A-->C) was found, whereas 1 subject carried the homozygous substitution that codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X(7) on lymphocytes was not affected by this E496A polymorphism, demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the E496A homozygote subject expressed nonfunctional receptor, whereas heterozygotes showed P2X(7) function that was half that of germline P2X(7). Results of transfection experiments showed that the mutant P2X(7) receptor was nonfunctional when expressed at low receptor density but regained function at a high receptor density. This density dependence of mutant P2X(7) function was also seen on differentiation of fresh monocytes to macrophages with interferon-gamma, which up-regulated mutant P2X(7) and partially restored its function. P2X(7)-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X(7) compared with germline (8.6 versus 35.2%). The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X(7) receptor.

Alanine↗
PubMed2001

Detection of P2X purinergic receptors on human B lymphocytes.

B lymphocytes are known to synthesise the P2X7 subtype of the P2X purinergic receptor family; however, the identification of the other six P2X subtypes on these cells has been limited by the absence of specific antibodies. In this study, we used a panel of anti-P2X polyclonal antibodies and confocal microscopy to examine the presence of each P2X receptor on human B lymphocytes. We observed that P2X1, P2X2, P2X4 and P2X7 subtypes, but not P2X3, P2X5 and P2X6 subtypes, are present on B lymphocytes.

Adenosine Triphosphate↗
PubMed2000

Expression of P2X(7) purinoceptors on human lymphocytes and monocytes: evidence for nonfunctional P2X(7) receptors.

Lymphocytes from normal subjects and patients with B-chronic lymphocytic leukemia (B-CLL) show functional responses to extracellular ATP characteristic of the P2X(7) receptor (previously termed P2Z). These responses include opening of a cation-selective channel/pore that allows entry of the fluorescent dye ethidium and activation of a membrane metalloprotease that sheds the adhesion molecule L-selectin. The surface expression of P2X(7) receptors was measured in normal leucocytes, platelets, and B-CLL lymphocytes and correlated with their functional responses. Monocytes showed four- to fivefold greater expression of P2X(7) than B, T, and NK lymphocytes, whereas P2X(7) expression on neutrophils and platelets was weak. All cell types demonstrated abundant intracellular expression of this receptor. All 12 subjects with B-CLL expressed lymphocyte P2X(7) at about the same level as B lymphocytes from normal subjects. P2X(7) function, measured by ATP-induced uptake of ethidium, correlated closely with surface expression of this receptor in normal and B-CLL lymphocytes and monocytes (n = 47, r = 0.70; P < 0.0001). However, in three patients the ATP-induced uptake of ethidium into the malignant B lymphocytes was low or absent. The lack of P2X(7) function in these B lymphocytes was confirmed by the failure of ATP to induce Ba(2+) uptake into their lymphocytes. This lack of function of the P2X(7) receptor resulted in a failure of ATP-induced shedding of L-selectin, an adhesion molecule that directs the recirculation of lymphocytes from blood into the lymph node.

Adenosine Triphosphate↗
PubMed1999

Activation of the P2Z/P2X7 receptor in human lymphocytes produces a delayed permeability lesion: involvement of phospholipase D.

Leukemic lymphocytes possess a cytolytic P2Z/P2X7 receptor which, when activated by extracellular ATP, opens a Ca2+- and Ba2+-permeable ion channel. This ATP-stimulated influx of divalent cations has been shown to activate an intracellular phospholipase D (PLD) which hydrolyzes membrane phosphatidylcholine. Lymphocytes that were exposed to ATP for 20 min at 37 degrees C, washed, and then incubated without ATP for 2 h showed an increased uptake of propidium2+, a dye widely used to measure cytotoxicity. The potent P2Z/P2X7 receptor inhibitor, KN-62, which is known to prevent the channel opening when added with ATP, did not block development of the permeability lesion when added 15 min before dye addition. The activity of lymphocyte PLD was stimulated fourfold by ATP and a proportion of this increased activity persisted for several hours after removal of ATP. Loading lymphocytes with intracellular choline+ by prior incubation of cells with ATP in isotonic choline chloride abolished both ATP-stimulated PLD activity and the ATP-induced permeability lesion. Addition of PLD but not phospholipase C to the extracellular medium increased lymphocyte permeability to propidium2+ and this effect was not observed in a choline medium. The cytolytic effect of exogenous PLD together with the inhibitory effect of choline, a product of the PLD reaction, suggests that sustained activation of intracellular PLD may be involved in the ATP-initiated cytolytic pathway.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1999

Transendothelial migration of lymphocytes in chronic lymphocytic leukaemia is impaired and involved down-regulation of both L-selectin and CD23.

Chronic lymphocytic leukaemia (B-CLL) is characterized by a progressive accumulation of B lymphocytes in blood and bone marrow and high concentrations of soluble CD23 and L-selectin are found in the serum of these patients. In this study lymphocytes from normal subjects and patients with B-CLL were allowed to undergo transendothelial migration across confluent layers of human umbilical vein endothelial cells. Lymphocytes in B-CLL samples showed an impaired capacity to migrate while the minor proportion of normal T cells was enriched by a mean of 2.5-fold in the transmigrated lymphocytes. In contrast, the ratio of B to T lymphocytes in normal preparations was unchanged in the transmigrated population. The expression of adhesion molecules on B-CLL lymphocytes before and after transendothelial migration was studied by flow cytometry which showed that 71 +/- 5% of L-selectin was lost from the surface of transmigrated lymphocytes. T and B cells from normal subjects also showed a major loss of L-selectin after transmigration. B-CLL lymphocytes and normal B cells expressed CD23 but this molecule was down-regulated following transendothelial migration, whereas the expression of VLA-4, ICAM-1, LFA-1 and CD44 was unchanged. Lymphocytes incubated with oxidized ATP, an irreversible inhibitor of P2Z/P2X7 purinoceptors, retained their capacity for transendothelial migration and showed the same loss of L-selectin as control leukaemic lymphocytes. Our results show that B-CLL lymphocytes have impaired ability for transendothelial migration compared to normal peripheral blood lymphocytes. Moreover, transendothelial migration involves a universal loss of L-selectin and CD23 from lymphocytes which suggests that the high serum levels of soluble L-selectin and CD23 observed in B-CLL may be generated by shedding during the process of transendothelial migration.

B-Lymphocytes↗
PubMed1999

P2Z purinoceptor, a special receptor for apoptosis induced by ATP in human leukemic lymphocytes.

OBJECTIVE: To investigate the role of purinergic P2Z receptors for apoptosis of human leukemic lymphocytes mediated by extracellular adenosine triphosphate (ATP). METHODS: A total of 13 B-chronic lymphocytic leukemia (CLL) patients were studied. Exposure of leukemic lymphocytes with (n = 8) or without (n = 5) P2Z receptors to ATP, benzoylbenzoic-ATP (BzATP), 2-methylthio-ATP (2MeSATP), adenosine-5' [gamma-thio] triphosphate (ATP-gamma S), and other nucleosides for 8 h in vitro. Apoptosis was detected by electron microscopy (EM), agarose gel electrophoresis, and the quantitative assay-TdT assay. RESULTS: Apoptosis was detected only in leukemic lymphocytes with P2Z receptors. Using a quantitative assay, ATP-induced DNA strand breaks were found to occur specifically with BzATP, ATP and 2MeSATP, but not for analogue ATP-gamma S nor other nucleosides. Meanwhile, ATP-induced DNA fragmentation was fully blocked by pretreatment with oxidized ATP (OxATP), a compound recently shown to block P2Z receptors. Also, it is shown that the Ca2+/calmodulin complex plays a role in the regulation of the apoptosis induced by ATP on CLL cells, because an antagonist of this complex, 1-[N, O-bis (5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) was found to inhibit the ATP-induced apoptosis. Furthermore, choline, an inhibitor of phospholipase D (PLD), is first shown to partially inhibit ATP-induced apoptosis. CONCLUSION: These data indicate that P2Z receptors on lymphocytes play an important role in the apoptosis induced by ATP in vitro.

Adenosine Triphosphate↗
PubMed1999

Role of bivalent cations and choline in ATP-induced apoptosis of human lymphocytes with P2Z receptors.

The role of bivalent cations and choline in ATP-induced apoptosis via P2Z purinoceptor was investigated in human leukemic lymphocytes. In vitro exposure of leukemic lymphocytes with P2Z receptors to 1 mmol/L ATP or 0.1 mmol/L benzoylbenzoic ATP (BzATP) for 8 h in the presence of choline, 1 mmol/L Mg2+ or other bivalent cations, and ATP-induced DNA breaks, associated with apoptosis were quantified by TdT assay. We observed that (1) Extracellular Mg2+ or Ca2+ stimulated ATP-induced DNA fragmentation in a dose-dependent manner, and the compatible evidence was provided by the inhibition of ATP-induced DNA fragmentation in the present of EGTA or EDTA; (2) ATP-induced DNA fragmentation was completely inhibited by 1 mmol/L Zn2+; (3) ATP-induced DNA breaks were not affected by Ba2+, Sr2+, Co2+ when they were substituted for extracellular Mg2+ or Ca2+; (4) Choline, an inhibitor of phospholipase D (PLD) stimulated by ATP through P2Z receptor in human lymphocytes, was also a partial inhibitor of ATP-induced DNA fragmentation, and the results were confirmed by flow cytometric analysis (FCA); (5) ATP-induced DNA fragmentation was completely obliterated when the temperature was lower than 10 degrees C. These results suggest that the endonuclease and PLD may be involved in ATP-induced apoptosis in human lymphocytes via P2Z receptor.

Adenosine Triphosphate↗
PubMed1998

Adenosine triphosphate-induced shedding of CD23 and L-selectin (CD62L) from lymphocytes is mediated by the same receptor but different metalloproteases.

CD23 is a transmembrane protein expressed on the surface of B-lymphocytes that binds IgE, CD21, CD11b, and CD11c. High concentrations of soluble CD23 and L-selectin are found in the serum of patients with B-chronic lymphocytic leukemia (B-CLL). Because extracellular adenosine triphosphate (ATP) causes shedding of L-selectin via activation of P2Z/P2X7 receptors expressed on B-CLL lymphocytes, we studied the effect of ATP on shedding of CD23. ATP-induced shedding of CD23 at an initial rate of 12% of that for L-selectin, whereas the EC50 for ATP was identical (35 micromol/L) for shedding of both molecules. Furthermore, benzoylbenzoyl ATP also produced shedding of CD23 and L-selectin with the same agonist EC50 values for both (10 micromol/L). Inactivation of the P2Z/P2X7 receptor by preincubation with oxidized ATP abolished ATP-induced shedding of both molecules. Moreover, KN-62, the most potent inhibitor for the P2Z/P2X7 receptor, inhibited ATP-induced shedding of both CD23 and L-selectin with the same IC50 (12 nmol/L). Ro 31-9790, a membrane permeant zinc chelator that inhibits the phorbol-ester-stimulated shedding of L-selectin, also inhibited shedding of CD23 from B-CLL lymphocytes. However, the IC50 for this inhibition by Ro31-9790 was different for L-selectin and CD23 (83 v 6 micromol/L, respectively). Although L-selectin was completely shed by incubation of cells with phorbol-ester, CD23 was not lost under these conditions. The data show that extracellular ATP induces shedding of L-selectin and CD23 from B-CLL lymphocytes by an action mediated by the P2Z/P2X7 receptor. However, different membrane metalloproteases seem to mediate the shedding of L-selectin and CD23.

Adenosine Triphosphate↗
PubMed1998

Flow cytometric studies of nucleoside transport regulation in single chromaffin cells.

The present paper reveals that a fluorescent derivative of nitrobenzylthioinosine, 5-(SAENTA-x8)-fluorescein, is a highly specific inhibitor of the neural NBTI-sensitive nucleoside transporter. 5-(SAENTA-x8)-fluorescein inhibited adenosine transport and [3H]NBTI binding with a Ki of 4 nM in cultured chromaffin cells. Flow cytometry demonstrated that 5-(SAENTA-x8)-fluorescein specifically interacted with the NBTI-sensitive nucleoside transporters with high affinity (K[D] = 6 nM). Activation of protein kinases A and C with forskolin or nicotinic receptor agonists, respectively, resulted in 50% inhibition of the fluorescence bound to the cells. Flow cytometry will allow studying nucleoside transport in single cells from heterogeneous neural cell populations.

Adenosine↗
PubMed1998

Partial agonists and antagonists reveal a second permeability state of human lymphocyte P2Z/P2X7 channel.

Extracellular ATP is known to trigger apoptosis of thymocytes and lymphocytes through a P2Z receptor at which ATP is a partial agonist, giving only 70% of the maximum response of 3'-O-(4-benzoyl)benzoyl-adenosine 5'-triphosphate (BzATP), a full agonist. This cytolytic receptor and its associated ion channel are Ca2+ (and Ba2+) selective but also pass molecules up to the size of ethidium cation (314 Da). RT-PCR showed identity between lymphocyte P2Z and the hP2X7 gene recently cloned from human monocytes. When human leukemic B lymphocytes were incubated with ATP and 133Ba2+, an immediate influx of isotope occurred. It was augmented by 45% when ATP was added 10 min before isotope. Time-resolved flow cytometry was used to examine kinetics of ethidium uptake in cells incubated with BzATP or the partial agonists ATP, 2-methylthioadenosine 5'-triphosphate, or adenosine 5'-O-(3-thiotriphosphate). Maximally effective concentrations of BzATP (50 microM) induced immediate uptake of ethidium at a rate linear with time. In contrast, a delay was observed (30 s) before ethidium uptake commenced after addition of maximally effective ATP concentrations (500 microM) at 37 degreesC, and the delay was longer at 24 degreesC. ATP addition 2-10 min before ethidium abolished the delay. The delay was longer with other partial agonists and inversely related to maximal flux produced by agonist. A delay was also observed for submaximal BzATP concentrations (10-20 microM). P2Z/P2X7 inhibitors, KN-62 and 5-(N, N-hexamethylene)-amiloride, reduced the rate of agonist-induced ethidium uptake and lengthened the delay. The results support a model in which agonists for P2Z/P2X7 receptor mediate an immediate channel opening allowing passage of small inorganic cations, followed by a slow further permeability increase allowing passage of larger permeant cations like ethidium. The rate of the second step depends on time and temperature and the efficacy and concentration of agonist and is slowed by antagonists, suggesting it depends on the fraction of P2Z/P2X7 channels held in the initial open state.

Adenosine Triphosphate↗
PubMed1998

[Apoptosis of leukemic lymphocytes mediated by purinergic P2z receptors].

OBJECTIVE: To investigate the role of purinergic P2z receptors for apoptosis of human leukemic lymphocytes induced by extracellular adenosine triphosphate (ATP). METHODS: A total of 11 B-CLL patients were studied with regard to exposure of leukemic lymphocytes with (n = 8) or without (n = 3) P2z receptors to ATP, benzoylbenzoic-ATP (BzATP), 2-methylthio-ATP (2MeSATP), adenosine-5'-[gamma-thio] triphosphate (ATP-gamma S), and other nucleosides for 8 h in vitro. Apoptosis was detected by electron microscopy (EM), agarose gel electrophoresis, and quantitative assay-TdT assay. RESULTS: Apoptosis was detected only in leukemic lymphocytes with P2z receptors. Using a quantitative assay, we found that ATP-induced DNA strand breaks occur specifically with BzATP, ATP and 2MeSATP, but not for analogue ATP-gamma S nor other nucleosides. Meanwhile, ATP-induced DNA fragmentation was fully blocked by pretreatment with oxidized ATP (OxATP), a compound recently shown to block P2z receptors. Also, the Ca2+/calmodulin complex played a role in the regulation of the apoptosis induced by ATP on CLL cells, because an antagonist of this complex, 1-[N,O-bis (5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) was found to inhibit the ATP-induced apoptosis. CONCLUSION: These data indicate that P2z receptors on lymphocytes play an important role in apoptosis induced by ATP in vitro.

Adenosine Triphosphate↗
PubMed1998

[Study on apoptosis of leukemic lymphocytes mediated by purinergic P2z receptors].

OBJECTIVE: To investigate the role of purinergic P2z receptors in human leukemic lymphocyte apoptosis induced by extracellular adenosine triphosphate (ATP). METHODS: Eleven B-CLL patients were studied. Leukemic lymphocytes with (n = 8) or without (n = 3) P2z receptors were exposed in vitro to ATP, benzoyl-benzoic-ATP (BzATP), 2-methylthio-ATP(2MeSATP), adenosine-5'-[gamma-thio] triphosphate (ATP-gamma S), and other nucleosides for 8 h. Apoptosis was detected by electron microscopy (EM), agarose gel electrophoresis, and quantitative TdT assay. RESULTS: Apoptosis was detected only in leukemic lymphocytes with P2z receptors. By using the quantitative assay, ATP-inducing DNA strand breaks were found to occur specifically for BzATP, ATP and 2MeSATP, but not for ATP-gamma S and other nucleosides. Meanwhile, ATP-inducing DNA fragmentation was fully blocked by pretreatment with oxidized ATP (OxATP), a compound recently shown to block P2z receptors. Ca2+/Calmodulin complex played a role in the regulation of the CLL cell apoptosis induced by ATP, because an antagonist of this complex, 1-[N,O-bis (5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), was found to inhibit the ATP-inducing apoptosis. CONCLUSION: P2z receptors on lymphocytes play an important role in the apoptosis induced by ATP in vitro.

Adenosine Triphosphate↗