HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Saunders, B. M. Articles by Wiley, J. S. Search for Related Content PubMed PubMed Citation Articles by Saunders, B. M. Articles by Wiley, J. S. Pubmed/NCBI databases Gene GEO Profiles HomoloGene OMIM UniGene Compound via MeSH Substance via MeSH

A Loss-of-Function Polymorphism in the Human P2X₇ Receptor Abolishes ATP-Mediated Killing of Mycobacteria ¹

Bernadette M. Saunders^2,*,, Suran L. Fernando, Ronald Sluyter, Warwick J. Britton^*, and James S. Wiley

^* Centenary Institute of Cancer Medicine and Cell Biology, Newtown, NSW Australia; Department of Medicine, University of Sydney, Sydney, New South Wales, Australia; and Department of Medicine, Nepean Hospital, University of Sydney, Penrith, NSW, Australia

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Protective immunity to mycobacterial infections requires activation of the antibacterial mechanisms of infected macrophages. It has previously been reported that ATP treatment of mycobacteria-infected macrophages induces apoptosis mediated via the P2X₇ pathway and that this leads to the death of both the host cell and the internalized bacilli. We have recently identified a single nucleotide polymorphism in the P2X7 gene (1513AC), with 1–2% prevalence in the homozygous state, which codes for a nonfunctional receptor. IFN--primed, mycobacteria-infected macrophages from wild-type individuals were incubated with ATP and this induced apoptosis and reduced mycobacterial viability by 90%. Similar treatment of macrophages from individuals homozygous for the 1513C polymorphism failed to induce apoptosis and did not lead to mycobacterial killing via the P2X₇-mediated pathway. These data demonstrate that a single nucleotide polymorphism in the P2X7 gene can allow survival of mycobacteria within infected host cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Tuberculosis (TB) ³ remains an enormous global health problem. The most recent World Health Organization figures estimate that 32% of the world’s population are infected with Mycobacterium tuberculosis. Disease from M. tuberculosis is most commonly caused, not by primary infection, but by the reactivation of dormant infection that the patient may have carried for many years. Factors that control progression of disease are still poorly defined for the majority of cases reported. HIV infection is the strongest risk factor for activation of disease, but the majority of non-HIV-infected patients do not have clear risk factors associated with disease progress. Racial variation in the susceptibility to TB and studies in twins strongly suggest that genetic factors are important (1). Mendelian inheritance, autosomal recessive mutations in genes for IFN-, IL-12, and STAT-1 pathway are rare causes for increased susceptibility to Mycobacterium bovis bacillus Calmette-Guérin (BCG) and Mycobacterium avium infection (2, 3). However, the major genetic effect is polygenic, and identified genetic risk factors, such as HLA type, mutations in the vitamin D₃ receptor, and polymorphisms in the NRAMP1 gene (1, 4, 5), only account for a minor portion of the increased susceptibility to M. tuberculosis.

Control of TB requires the generation of Ag-specific T cell responses, the activation of infected macrophages, and formation of granulomatous lesions to wall off infected macrophages and prevent dissemination of the bacilli. Macrophages play multiple roles during mycobacterial infection. They are the principal host cells for intracellular replication of these bacteria, act as APCs during reactivation of lymphocytes at the sites of infection, and are responsible for killing of mycobacteria (6).

Extracellular ATP can induce macrophage bacteriocidal activity toward mycobacteria. ATP initiates both macrophage apoptosis and killing of the intracellular mycobacteria in infected macrophages. The killing of mycobacteria by humans is independent of reactive nitrogen and oxygen intermediates (7). ATP exerts its bacteriocidal activity through activation of the P2X₇ purinergic receptor (8), which is highly expressed on human and murine macrophages and is further up-regulated by IFN- (9, 10, 11).

P2X₇ receptors are ligand-gated cation channels with two transmembrane domains and a trimeric structure in the plasma membrane (12, 13). Activation of P2X₇ causes an immediate opening of a cation-selective channel, which undergoes dilatation within seconds to allow entry of larger cations up to the size of ethidium (12, 14). Activation of the P2X₇ receptor stimulates a number of downstream events, the most important of which is the triggering of the caspase cascade, leading to apoptotic death of the target cell (15, 16).

We have recently described a single nucleotide polymorphism (1513AC) in the P2X7 gene which changes the glutamic acid to alanine at aa 496 and abolishes P2X₇ function without affecting expression of the receptor on lymphocytes (10). The frequency of the 1513C allele is 0.12 in the Caucasian population and this yields an expected prevalence of the homozygous condition of 1–2% (17, 18).

Treatment of mycobacteria-infected macrophages with ATP induces apoptosis, mediated via the P2X₇ pathway, and leads to the death of both the host cell and the internalized bacilli. We have measured mycobacterial killing and apoptotic death of infected macrophages from three individuals homozygous for the loss-of-function polymorphic allele. These data demonstrate a complete loss of ATP-induced mycobacterial killing in these individuals.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Subject recruitment

A total of 115 adult subjects of Caucasian background were screened for P2X7 genotype to identify three who were homozygous for the 1513AC allele (10). Blood was taken with informed consent (and ethics approval) from the 18 subjects described in this study.

Human monocytes and monocyte-derived macrophage cultures

PBMC were separated on Ficoll-Paque PLUS (Amersham Pharmacia Biotech, Uppsala, Sweden) and used to provide fresh monocytes. To generate monocyte-derived macrophages, PBMC were resuspended at 1–2 x 10⁶ cells/ml in RPMI 1640 (Sigma-Aldrich, St. Louis, MO) containing 10% heat-inactivated FCS (Life Technologies, Auckland New Zealand) and 2 mM glutamine (Sigma-Aldrich). Cells were incubated for 2 h and washed twice to remove nonadherent cells. Monocytes (adherent cells) were cultured for 6 days in complete medium. For ATP-induced ethidium uptake and P2X₇ expression measurements, 6-day macrophages were cultured overnight with IFN- (100 IU/ml; Roche, Sydney, Australia) and collected by mechanical scraping. For mycobacterial killing and apoptosis assays, 6-day macrophages were detached and plated in 96-well plates at 1 x 10⁵ cells/well in antibiotic-free medium with IFN- (100 IU/ml) overnight, before mycobacterial infection.

Ethidium influx measurement by flow cytometry

PBMC or IFN--activated macrophages, prelabeled with FITC-conjugated anti-human CD14 mAb (DAKO, Carpinteria, CA), were resuspended in 1 ml of HEPES-buffered KCl medium (10 mM HEPES (Life Technologies), 150 mM KCl, 5 mM D-glucose, and 1% BSA (Sigma-Aldrich), pH 7.5) at 37°C. All samples were stirred and temperature controlled at 37°C. Ethidium⁺ (25 µM; Sigma-Aldrich) was added, followed 40 s later by addition of 1 mM ATP (Sigma-Aldrich). Cells were analyzed at 1000 events on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). The linear mean channel of fluorescence intensity for gated CD14⁺ cells over successive 5-s intervals was analyzed by WinMDI software version 2.7 (available at http://facs.scripps.edu/software.html) and plotted against time. Because of the increased P2X₇ function on macrophages, data for macrophages were acquired at a reduced voltage setting for FL-2 (ethidium fluorescence) as described previously (19).

Immunofluorescent staining and flow cytometry

PBMC or IFN--activated macrophages were labeled with FITC-conjugated anti-human P2X₇ (20) or isotype control mAb (DAKO), PE-conjugated anti-human CD14 mAb (DAKO) and 7-amino actinomycin D (Sigma-Aldrich), or alone with PerCP-conjugated anti-human HLA-DR mAb (BD Biosciences) in the presence of 10% human AB serum for 20 min, washed, and analyzed using a FACSCalibur flow cytometer.

Mycobacterial killing and apoptosis

IFN--activated macrophages were infected with BCG-green fluorescent protein (21) for 4 h at a multiplicity of infection of 5:1 (day 0), then washed twice to remove extracellular bacteria. On day 2, cells were pulsed with 3 mM ATP for 20 min, washed, and incubated overnight (7). On day 3, one-half of the wells were lysed with 0.1% Triton-X for 30 min to release viable bacilli. Serial dilutions of cell lysates were plated onto 7H11 agar and incubated at 37°C for 3–4 wk to determine the load of viable mycobacteria. The remaining wells were stained for PE-conjugated annexin V and propidium iodide (PI) to measure apoptosis as per the manufacturer’s protocol (BD Biosciences) and analyzed on a FACSCalibur flow cytometer.

DNA extraction, PCR, and DNA sequencing

Genomic DNA was extracted from peripheral blood using the Wizard Genomic DNA Purification kit (Promega, Madison, WI). A primer pair within exon 13 of the P2X7 gene amplified a 376-bp product from genomic DNA. The forward primer was 5'-ACTCCTAGATCCAGGGATAGCC-3' and the reverse primer was 5'-TACAGACGTGAGCCACGGT-3'. PCR amplification (25 cycles of denaturation at 95°C for 45 s, annealing at 56°C, and extension at 72°C for 1 min). PCR products were separated in 2% agarose gel and visualized by ethidium bromide staining. Amplified PCR products were purified using the GFX PCR DNA and Gel Band Purification kit (Amersham Biosciences, Piscataway, NJ) and sequenced using an ABI PRISM 377 DNA sequencer (Applied Biosystems, Foster City, CA).

Statistics

Ethidium uptake and P2X₇ expression data were analyzed by the unpaired Student’s t test. Mycobacterial killing and apoptosis data were analyzed by the Mann-Whitney U test. Data were considered significant when p < 0.001.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
P2X₇ expression and function in monocytes

The functional activity of the P2X₇ receptor was investigated in three individuals homozygous for the 1513C single nucleotide polymorphism. Monocytes from these individuals were treated with ATP and ethidium uptake was measured (Fig. 1). Unlike wild-type individuals, all three homozygous subjects displayed no detectable uptake above basal levels. P2X₇ function was quantitated by measurement of the arbitrary units of area under the ethidium uptake curve; this was 19393 ± 1803 (n = 16) for the wild-type monocytes compared with 73 ± 73 (n = 3, subjects tested at least twice) for monocytes from the 1513C homozygous subjects (p < 0.001). The surface expression of P2X₇ on monocytes from wild-type individuals (37.5 ± 6.1, n = 11) was also higher than the P2X₇ expression on monocytes from homozygous individuals (23.9 ± 11.2, n = 3); however, this difference did not reach significance (p = 0.29).

View larger version (22K): [in this window] [in a new window]   FIGURE 1. ATP induced ethidium uptake curve. PBMC (2 x 106) from a wild-type individual (A) and an individual homozygous for the 1513AC polymorphism (B) were labeled with anti-CD14-FITC conjugate and incubated in HEPES-buffered KCl at 37°C. Ethidium bromide (25 µM) was added and followed 40 s later by 1 mM ATP. The mean channel of cell-associated fluorescence intensity was measured at 5-s intervals. Data are representative of wild-type individuals and those with the 1513AC polymorphism. Ethidium uptake in the absence (•) or presence of ATP ().

ATP-induced ethidium uptake and P2X₇ expression were also measured in IFN--activated macrophages. ATP-induced ethidium uptake and P2X₇ expression were 18- and 12-fold higher, respectively, in wild-type macrophages than homozygous macrophages (Table I). However, this single nucleotide polymorphism did not effect the expression of class II molecules because we saw similar levels of HLA-DR expression (Table I). Further BCG infection induced increased TNF secretion by all macrophages regardless of their P2X₇ phenotype (Fig. 2). Although the level of TNF produced varied among individuals, no significant difference was seen in TNF induction between macrophages from wild-type and homozygote individuals.

View larger version (10K): [in this window] [in a new window]   FIGURE 2. TNF induction in BCG-infected human macrophages. Human monocyte-derived macrophages were preincubated with 100 U/ml IFN- 16 h before infection with BCG-green fluorescent protein (5:1 multiplicity of infection). After 4 h, extracellular bacteria were removed by washing and cells were incubated for an additional 48 h. One-half of the cells were then pulsed for 20 min with 3 mM ATP, washed, and incubated for another 16 h. Supernatant was collected and TNF production was measured by ELISA. Data are TNF production by infected macrophages, without ATP pulsing, from three wild-type and three homozygous individuals. TNF production by uninfected macrophages was 39.4 ± 12.3 pg from wild-type subjects and 66.9 ± 26.7 pg from homozygotes.

It has previously been shown that ATP-induced apoptosis, but not H₂O₂-induced necrosis, of mycobacteria-infected macrophages leads to the death of the intracellular bacilli (22). We investigated whether the loss-of-function polymorphism at 1513 of the P2X7 gene affects the ability of phagocytic cells to kill mycobacteria via ATP-mediated apoptosis. Adherent monocyte-derived macrophages were infected with M. bovis BCG for 48 h and then pulsed with ATP for 20 min. Following overnight incubation, the percentage of apoptotic cells and the number of viable bacilli were determined. ATP treatment of BCG-infected macrophages from 1513C homozygote subjects failed to increase the number of apoptotic macrophages (-0.3–5.7%; Fig. 3), whereas in wild-type individuals ATP treatment of infected macrophages led to a 10-fold increase in the level of apoptosis (19.8–31.2%; Fig. 3). The level of necrosis (measured as PI⁺/annexin^- macrophages) was equivalent in both groups, 0.29–5.25 for wild-type individuals and 0.11–3.01 for homozygous individuals. In wild-type macrophages, this increase in apoptosis was associated with a 90% reduction in mycobacterial load (Fig. 4). By contrast, the bacillary load in homozygous macrophages was unchanged after ATP treatment (Fig. 4).

View larger version (11K): [in this window] [in a new window]   FIGURE 3. ATP induced apoptosis in BCG-infected human macrophages. Human monocyte-derived macrophages were infected for 48 h as described in Fig. 2. Cells were then pulsed for 20 min with 3 mM ATP, washed, and incubated for an additional 16 h. Cells were stained with annexin V and PI. Cells were identified as early apoptotic (annexin+/PI-), late stage apoptotic (annexin+/PI+), or necrotic (annexin-/PI+). Data are the percentage of apoptotic cells (early and late stage) in ATP-pulsed cultures minus non-ATP-pulsed cultures in three individuals homozygous for the 1513AC polymorphism and in three wild-type individuals tested on two separate occasions. Differences between wild-type individuals and individuals homozygous for the 1513AC polymorphism were significant at *, p < 0.001.

View larger version (12K): [in this window] [in a new window]   FIGURE 4. ATP induced killing of BCG within human macrophages. Human monocyte-derived macrophages were infected for 48 h as described in Fig. 2. Cells were then pulsed for 20 min with 3 mM ATP, washed, and incubated for another 16 h. Cells were lysed and viable bacilli were enumerated by plating serial dilutions of cell lysates onto 7H11 agar. Data shows the log reduction in viable bacilli in ATP-pulsed cultures minus non-ATP-pulsed cultures in three individuals homozygous for the 1513AC polymorphism and in three wild-type individuals tested on two separate occasions. Differences between wild-type individuals and individuals homozygous for the 1513AC polymorphism were significant at *, p < 0.001.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

There is increasing evidence that P2X₇ plays an essential role in mycobacterial killing by cells of the monocyte/macrophage lineage. Extracellular ATP, added to infected human macrophages, has been shown to induce the apoptotic death of the host cell and its internalized mycobacteria (7, 23). This effect of ATP is known to be mediated by P2X₇, as ATP-induced apoptosis and subsequent bacterial killing were inhibited by treatment of infected macrophages with the P2X₇ antagonist KN62 (7, 24). Furthermore, macrophages from P2X7 gene knockout mice were unable to affect killing of mycobacteria by the addition of ATP (25).

The mechanism of P2X₇-mediated killing involves a transient increase in cytosolic calcium which is necessary for the fusion of lysosomes with infected phagosomes and subsequent mycobacterial killing within the phagolysosome (23, 26). ATP-mediated killing of BCG within Nramp-susceptible, p47^phox-/- and INOS^-/- mice was unaffected, demonstrating that the pathway of ATP killing is independent of reactive oxygen intermediates, reactive nitrogen intermediates, and Nramp (25). Another downstream effect of P2X₇ receptor activation is to stimulate the activity of phospholipase D, which is required for the killing of intracellular mycobacteria (8).

ATP-induced apoptosis and ATP-mediated killing of mycobacteria were essentially absent in macrophages from homozygous subjects despite ATP inducing a low level of ethidium uptake into these cells. Previously, we have observed marked differences in the ATP-induced function of macrophages from homozygotes compared with wild-type individuals as measured by ethidium uptake (10). These differences in channel function were associated with marked differences in biological function, as measured by apoptosis and mycobacterial killing. ATP-induced apoptosis of macrophages is dependent on caspase activation (16), while ATP-mediated killing of mycobacteria requires the stimulation of phospholipase D to cause phagosome-lysosome fusion (23, 25). Phospholipase D stimulation, however, is not necessary for ATP-induced macrophage death (8, 25) or ATP-induced pore formation (27). Therefore, the requirements for pore formation per se may be much simpler than those needed for apoptosis or killing of intracellular mycobacteria, and thus these latter events may be more sensitive to loss-of-function mutations in P2X₇.

Our data suggest that loss-of-function polymorphisms in the P2X7 gene may confer a genetic susceptibility to tuberculosis. Recently, a significant association against TB was found for a single nucleotide polymorphism for the upstream region of the P2X7 gene at nucleotide position -762 (28). Whether this polymorphism lies in the promoter region of P2X7 is uncertain and its effect on P2X₇ function is unknown. A recent study of nine healthy individuals failed to find an association between the 1513C allele and cytotoxic response to ATP (29). This study, however, looked at only one homozygote 1513C individual who, in agreement with our study, lacked responsiveness to ATP. Moreover, IFN- was a poor inducer of P2X₇ expression on macrophages from this individual (29), again consistent with our observations. We observed that P2X₇ expression was 12-fold lower on homozygous macrophages than on wild-type macrophages. Importantly in the study by Li et al. (29), macrophages from three of nine subjects who lacked responsiveness to ATP all had poor P2X₇ expression. Combined these results imply that low P2X₇ expression on macrophages caused by the 1513C allele in homozygous dosage, or some other defect, results in the inability of ATP to mediate killing of intracellular mycobacteria. Recently, a second loss-of-function polymorphism (1729TA) has been defined (19). This polymorphism is also in exon 13, which encodes the intracellular domain of P2X₇. This prevents normal trafficking of the P2X₇ receptor to the cell surface and reduces channel function. It will be of interest to determine whether this also results in impaired ATP-mediated killing of intracellular mycobacteria.

The overall impact of single nonfunctional polymorphisms, which affect macrophage activity, on TB susceptibility in a population depends on the gene frequency for that allele in the population studied. In the study on a Gambian population (28), there was a lower frequency (7.6%) of expression of the 1513AC polymorphism than the frequency of observed in this Australian Caucasian population (13.9%, n = 115 (Ref.10 and R.S. and J.S.W., unpublished observations). Therefore, this polymorphism may confer significant susceptibility to TB in this Caucasian population.

	Footnotes

 
¹ This work was supported by the National Health and Medical Research Council of Australia, the Community Health Anti Tuberculosis Association, the Cecilia Kilkeary Foundation, and the New South Wales Department of Health through its research infrastructure grant to the Centenary Institute of Cancer Medicine and Cell Biology. B.M.S. is a Rolf Edgar Lake Research Fellow of the Faculty of Medicine, University of Sydney.

² Address correspondence and reprint requests to Dr. Bernadette M. Saunders, Centenary Institute of Cancer Medicine and Cell Biology, Royal Prince Alfred Hospital, Locked Bag. No. 6, Newtown, 2042 NSW, Australia. E-mail address: b.saunders{at}centenary.usyd.edu.au

³ Abbreviations used in this paper: TB, tuberculosis; BCG, bacillus Calmette-Guérin; PI, propidium iodide.

Received for publication March 17, 2003. Accepted for publication September 9, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Bellamy, R., C. Ruwende, T. Corrah, K. P. McAdam, H. C. Whittle, A. V. Hill. 1998. Variations in the NRAMP1 gene and susceptibility to tuberculosis in West Africans. N. Engl. J. Med. 338:640.[Abstract/Free Full Text]
2. Doffinger, R., S. Dupuis, C. Picard, C. Fieschi, J. Feinberg, G. Barcenas-Morales, J. L. Casanova. 2002. Inherited disorders of IL-12- and IFN-mediated immunity: a molecular genetics update. Mol. Immunol. 38:903.[Medline]
3. Dupuis, S., E. Jouanguy, S. Al-Hajjar, C. Fieschi, I. Z. Al-Mohsen, S. Al-Jumaah, K. Yang, A. Chapgier, C. Eidenschenk, P. Eid, et al 2003. Impaired response to interferon-/ and lethal viral disease in human STAT1 deficiency. Nat. Genet. 33:388.[Medline]
4. Rook, G. A.. 1988. The role of vitamin D in tuberculosis. Am. Rev. Respir. Dis. 138:768.[Medline]
5. Cooke, G. S., A. V. S. Hill. 2001. Genetics of susceptibility to human infectious disease. Nat. Rev. Genet. 2:967.[Medline]
6. Schaible, U. E., H. L. Collins, S. H. Kaufmann. 1999. Confrontation between intracellular bacteria and the immune system. Adv. Immunol. 71:267.[Medline]
7. Lammas, D. A., C. Stober, C. J. Harvey, N. Kendrick, S. Panchalingam, D. S. Kumararatne. 1997. ATP-induced killing of mycobacteria by human macrophages is mediated by purinergic P2Z(P2X₇) receptors. Immunity 7:433.[Medline]
8. Kusner, D. J., J. Adams. 2000. ATP-induced killing of virulent Mycobacterium tuberculosis within human macrophages requires phospholipase D. J. Immunol. 164:379.[Abstract/Free Full Text]
9. Hickman, S. E., J. El Khoury, S. Greenberg, I. Schieren, S. C. Silverstein. 1994. P2Z adenosine triphosphate receptor activity in cultured human monocyte-derived macrophages. Blood 84:2452.[Abstract/Free Full Text]
10. Gu, B. J., W. Zhang, R. A. Worthington, R. Sluyter, P. Dao-Ung, S. Petrou, J. A. Barden, J. S. Wiley. 2001. A Glu-496 to Ala polymorphism leads to loss of function of the human P2X₇ receptor. J. Biol. Chem. 276:11135.[Abstract/Free Full Text]
11. Falzoni, S., M. Munerati, D. Ferrari, S. Spisani, S. Moretti, F. Di Virgilio. 1995. The purinergic P2Z receptor of human macrophage cells: characterization and possible physiological role. J. Clin. Invest. 95:1207.
12. Rassendren, F., G. Buell, A. Newbolt, R. A. North, A. Surprenant. 1997. The permeabilizing ATP receptor, P2X₇: cloning and expression of human cDNA. J. Biol. Chem. 272:5482.[Abstract/Free Full Text]
13. Nicke, A., H. G. Baumert, J. Rettinger, A. Eichele, G. Lambrecht, E. Mutschler, G. Schmalzing. 1998. P2X₁ and P2X₃ receptors form stable trimers: a novel structural motif of ligand-gated ion channels. EMBO J. 17:3016.[Medline]
14. Wiley, J. S., R. Chen, G. P. Jamieson. 1993. The ATP4-receptor-operated channel (P2Z class) of human lymphocytes allows Ba²⁺ and ethidium⁺ uptake-inhibition of fluxes by suramin. Arch. Biochem. Biophys. 305:54.[Medline]
15. Ferrari, D., M. Los, M. K. Bauer, P. Vandenabeele, S. Wesselborg, K. Schulze-Osthoff. 1999. P2Z purinoreceptor ligation induces activation of caspases with distinct roles in apoptotic and necrotic alterations of cell death. FEBS Lett. 447:71.[Medline]
16. Humphreys, B. D., J. Rice, S. B. Kertesy, G. R. Dubyak. 2000. Stress-activated protein kinase/JNK activation and apoptotic induction by the macrophage P2X₇ nucleotide receptor. J. Biol. Chem. 275:26792.[Abstract/Free Full Text]
17. Thunberg, U., G. Tobin, A. Johnson, O. Soderberg, L. Padyukov, M. Hultdin, L. Klareskog, G. Enblad, C. Sundstrom, R. Rosenquist. 2002. Polymorphism in the P2X₇ receptor gene and survival in chronic lymphocytic leukaemia. Lancet 360:1935.[Medline]
18. Wiley, J. S., L. P. Dao-Ung, B. J. Gu, R. Sluyter, A. N. Shemon, C. P. Li, J. Taper, J. Gallo, A. Manoharan. 2002. A loss-of-function polymorphic mutation in the cytolytic P2X₇ receptor gene and chronic lymphocytic leukaemia: a molecular study. Lancet 359:1114.[Medline]
19. Wiley, J. S., L. P. Dao-Ung, C. Li, A. N. Shemon, B. J. Gu, M. L. Smart, S. J. Fuller, J. A. Barden, S. Petrou, R. Sluyter. 2003. An Ile-568 to Asn polymorphism prevents normal trafficking and function of the human P2X₇ receptor. J. Biol. Chem. 278:17108.[Abstract/Free Full Text]
20. Buell, G. N., F. Talabot, A. Gos, J. Lorenz, E. Lai, M. A. Morris, S. E. Antonarakis. 1998. Gene structure and chromosomal localization of the human P2X₇ receptor. Recept. Channels 5:347.[Medline]
21. Triccas, J. A., F. X. Berthet, V. Pelicic, B. Gicquel. 1999. Use of fluorescence induction and sucrose counterselection to identify Mycobacterium tuberculosis genes expressed within host-cells. Microbiology 145:2923.[Abstract/Free Full Text]
22. Molloy, A., P. Laochumroonvorapong, G. Kaplan. 1994. Apoptosis, but not necrosis, of infected monocytes is coupled with killing of intracellular bacillus Calmette-Guerin. J. Exp. Med. 180:1499.[Abstract/Free Full Text]
23. Kusner, D. J., J. A. Barton. 2001. ATP stimulates human macrophages to kill intracellular virulent Mycobacterium tuberculosis via calcium-dependent phagosome-lysosome fusion. J. Immunol. 167:3308.[Abstract/Free Full Text]
24. Gargett, C. E., J. S. Wiley. 1997. The isoquinoline derivative KN-62 a potent antagonist of the P2Z-receptor of human lymphocytes. Br. J. Pharmacol. 120:1483.[Medline]
25. Fairbairn, I. P., C. B. Stober, D. S. Kumararatne, D. A. Lammas. 2001. ATP-mediated killing of intracellular mycobacteria by macrophages is a P2X₇-dependent process inducing bacterial death by phagosome-lysosome fusion. J. Immunol. 167:3300.[Abstract/Free Full Text]
26. Stober, C. B., D. A. Lammas, C. M. Li, D. S. Kumararatne, S. L. Lightman, C. A. McArdle. 2001. ATP-mediated killing of Mycobacterium bovis bacille Calmette-Guérin within human macrophages is calcium dependent and associated with the acidification of mycobacteria-containing phagosomes. J. Immunol. 166:6276.[Abstract/Free Full Text]
27. el-Moatassim, C., G. R. Dubyak. 1993. Dissociation of the pore-forming and phospholipase D activities stimulated via P2z purinergic receptors in BAC1.2F5 macrophages: product inhibition of phospholipase D enzyme activity. J. Biol. Chem. 268:15571.[Abstract/Free Full Text]
28. Li, C. M., S. J. Campbell, D. S. Kumararatne, R. Bellamy, C. Ruwende, K. McAdam, A. V. S. Hill, D. A. Lammas. 2002. Association of a polymorphism in the P2X₇ gene with tuberculosis in a Gambian population. J. Infect. Dis. 186:1458.[Medline]
29. Li, C. M., S. J. Campbell, D. S. Kumararatne, A. V. S. Hill, D. A. Lammas. 2002. Response heterogeneity of human macrophages to ATP is associated with P2X₇ receptor expression but not to polymorphisms in the P2RX7 promoter. FEBS Lett. 531:127.[Medline]

This article has been cited by other articles:

				L. Yip, T. Woehrle, R. Corriden, M. Hirsh, Y. Chen, Y. Inoue, V. Ferrari, P. A. Insel, and W. G. Junger Autocrine regulation of T-cell activation by ATP release and P2X7 receptors FASEB J, June 1, 2009; 23(6): 1685 - 1693. [Abstract] [Full Text] [PDF]

				L. C. Denlinger, L. Shi, A. Guadarrama, K. Schell, D. Green, A. Morrin, K. Hogan, R. L. Sorkness, W. W. Busse, and J. E. Gern Attenuated P2X7 Pore Function as a Risk Factor for Virus-induced Loss of Asthma Control Am. J. Respir. Crit. Care Med., February 15, 2009; 179(4): 265 - 270. [Abstract] [Full Text] [PDF]

				T. Noguchi, K. Ishii, H. Fukutomi, I. Naguro, A. Matsuzawa, K. Takeda, and H. Ichijo Requirement of Reactive Oxygen Species-dependent Activation of ASK1-p38 MAPK Pathway for Extracellular ATP-induced Apoptosis in Macrophage J. Biol. Chem., March 21, 2008; 283(12): 7657 - 7665. [Abstract] [Full Text] [PDF]

				S.-R. Woo, R. G. Barletta, and C. J. Czuprynski Extracellular ATP Is Cytotoxic to Mononuclear Phagocytes but Does Not Induce Killing of Intracellular Mycobacterium avium subsp. paratuberculosis Clin. Vaccine Immunol., September 1, 2007; 14(9): 1078 - 1083. [Abstract] [Full Text] [PDF]

				K.-H. Lee, S. S. Park, I. Kim, J. H. Kim, E. K. Ra, S.-S. Yoon, Y.-C. Hong, S. Park, and B. K. Kim P2X7 receptor polymorphism and clinical outcomes in HLA-matched sibling allogeneic hematopoietic stem cell transplantation Haematologica, May 1, 2007; 92(5): 651 - 657. [Abstract] [Full Text] [PDF]

				S. L. Fernando, B. M. Saunders, R. Sluyter, K. K. Skarratt, H. Goldberg, G. B. Marks, J. S. Wiley, and W. J. Britton A Polymorphism in the P2X7 Gene Increases Susceptibility to Extrapulmonary Tuberculosis Am. J. Respir. Crit. Care Med., February 15, 2007; 175(4): 360 - 366. [Abstract] [Full Text] [PDF]

				L. C. Denlinger, D. B. Coursin, K. Schell, G. Angelini, D. N. Green, A. G. Guadarrama, J. Halsey, U. Prabhu, K. J. Hogan, and P. J. Bertics Human P2X7 Pore Function Predicts Allele Linkage Disequilibrium Clin. Chem., June 1, 2006; 52(6): 995 - 1004. [Abstract] [Full Text] [PDF]

				G. Burnstock Pathophysiology and therapeutic potential of purinergic signaling. Pharmacol. Rev., March 1, 2006; 58(1): 58 - 86. [Abstract] [Full Text] [PDF]

				A. N. Shemon, R. Sluyter, S. L. Fernando, A. L. Clarke, L.-P. Dao-Ung, K. K. Skarratt, B. M. Saunders, K. S. Tan, B. J. Gu, S. J. Fuller, et al. A Thr357 to Ser Polymorphism in Homozygous and Compound Heterozygous Subjects Causes Absent or Reduced P2X7 Function and Impairs ATP-induced Mycobacterial Killing by Macrophages J. Biol. Chem., January 27, 2006; 281(4): 2079 - 2086. [Abstract] [Full Text] [PDF]

				G. Cabrini, S. Falzoni, S. L. Forchap, P. Pellegatti, A. Balboni, P. Agostini, A. Cuneo, G. Castoldi, O. R. Baricordi, and F. Di Virgilio A His-155 to Tyr Polymorphism Confers Gain-of-Function to the Human P2X7 Receptor of Human Leukemic Lymphocytes J. Immunol., July 1, 2005; 175(1): 82 - 89. [Abstract] [Full Text] [PDF]

				A. J. Myers, B. Eilertson, S. A. Fulton, J. L. Flynn, and D. H. Canaday The Purinergic P2X7 Receptor Is Not Required for Control of Pulmonary Mycobacterium tuberculosis Infection Infect. Immun., May 1, 2005; 73(5): 3192 - 3195. [Abstract] [Full Text] [PDF]

				R. Sluyter, A. N. Shemon, J. A. Barden, and J. S. Wiley Extracellular ATP Increases Cation Fluxes in Human Erythrocytes by Activation of the P2X7 Receptor J. Biol. Chem., October 22, 2004; 279(43): 44749 - 44755. [Abstract] [Full Text] [PDF]

				B. J. Gu, R. Sluyter, K. K. Skarratt, A. N. Shemon, L.-P. Dao-Ung, S. J. Fuller, J. A. Barden, A. L. Clarke, S. Petrou, and J. S. Wiley An Arg307 to Gln Polymorphism within the ATP-binding Site Causes Loss of Function of the Human P2X7 Receptor J. Biol. Chem., July 23, 2004; 279(30): 31287 - 31295. [Abstract] [Full Text] [PDF]

				L. C. Denlinger, K. Schell, G. Angelini, D. Green, A. Guadarrama, U. Prabhu, D. B. Coursin, K. Hogan, and P. J. Bertics A novel assay to detect nucleotide receptor P2X7 genetic polymorphisms influencing numerous innate immune functions Innate Immunity, April 1, 2004; 10(2): 137 - 142. [Abstract] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Saunders, B. M. Articles by Wiley, J. S. Search for Related Content PubMed PubMed Citation Articles by Saunders, B. M. Articles by Wiley, J. S. Pubmed/NCBI databases Gene GEO Profiles HomoloGene OMIM UniGene Compound via MeSH Substance via MeSH