Ben J. Gu

A loss-of-function polymorphic mutation in the cytolytic P2X7 receptor gene and chronic lymphocytic leukaemia: a molecular study.

BACKGROUND Chronic lymphocytic leukaemia (CLL) has a familial incidence nearly three times higher than expected for the general population and one predisposing factor might be an inherited failure of mechanisms involved in apoptosis of lymphocytes. Our aim was to ascertain whether or not a defect in a proapoptotic pathway, caused by a single nucleotide polymorphism that results in loss-of-function of P2X7 in healthy individuals, was present in leukaemic B lymphocytes of patients with CLL. METHODS We extracted genomic DNA from the peripheral blood leucocytes of 36 unrelated individuals with CLL, four individuals with familial CLL, and 46 age-matched controls. We sequenced a PCR product to detect mutations in exon 13 of P2X7. In most patients with CLL, we measured expression and function of the P2X7 receptor by flow cytometry in B lymphocytes and T lymphocytes. FINDINGS The prevalence of the polymorphic mutation and the frequency of the mutant allele were three-fold greater in individuals with CLL than in white, elderly controls. Individuals homozygous for the polymorphic allele had no P2X7 receptor function and heterozygotes had half the mean function of that seen in individuals homozygous for the wildtype allele; amounts of ATP-induced apoptosis varied accordingly. In two families, in which we studied a father-son pair and a sister-sister pair with CLL, loss of P2X7 function arose because of inheritance of one or two 1513A-->C alleles for P2X7. INTERPRETATION Activation of the P2X7 receptor leads to apoptosis of lymphocytes in individuals with CLL, and reduced function of this receptor has an anti-apoptotic effect, resulting in an increase in B-cell numbers. Thus, inheritance of a loss-of-function polymorphic mutation at position 1513 in the P2X7 gene could contribute to the pathogenesis of CLL.

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

The P2X7 receptor is a ligand-gated cation channel that is highly expressed on mononuclear leukocytes and that mediates ATP-induced apoptosis and killing of intracellular pathogens. There is a wide variation in P2X7 receptor function between subjects, explained in part by four loss-of-function polymorphisms (R307Q, E496A, I568N, and a 5′-intronic splice site polymorphism), as well as rare mutations. In this study, we report the allele frequencies of 11 non-synonymous P2X7 polymorphisms and describe a fifth loss-of-function polymorphism in the gene (1096C → G), which changes Thr357 to Ser (T357S) with an allele frequency of 0.08 in the Caucasian population. P2X7 function was measured by ATP-induced ethidium+ influx into peripheral blood lymphocytes and monocytes and, when compared with wild-type subjects, was reduced to 10–65% in heterozygotes, 1–18% in homozygotes, and 0–10% in compound heterozygotes carrying T357S and a second loss-of-function polymorphism. Overexpression of the T357S mutant P2X7 in either HEK-293 cells or Xenopus oocytes gave P2X7 function of ∼50% that of wild-type constructs. Differentiation of monocytes to macrophages, which also up-regulates P2X7, restored P2X7 function to near normal in cells heterozygous for T357S and to a value 50–65% of wild-type in cells homozygous for T357S or compound heterozygous for T357S/E496A. However, macrophages from subjects that are compound heterozygous for either T357S/R307Q or T357S/stop codon had near-to-absent P2X7 function. These functional deficits induced by T357S were paralleled by impaired ATP-induced apoptosis and mycobacteria killing in macrophages from these subjects. Lymphocytes, monocytes, and macrophages from subjects homozygous for T357S or compound heterozygous for T357S and a second loss-of-function allele have reduced or absent P2X7 receptor function.

The human P2X7 receptor and its role in innate immunity.

The human P2X7 receptor is a two-transmembrane ionotropic receptor which has a ubiquitous distribution and is most highly expressed on immune cells. In macrophages and similar myeloid cells primed by lipopolysaccharide (LPS), activation of P2X7 by extracellular ATP opens a cation channel/pore allowing massive K+ efflux associated with processing and secretion of pro-inflammatory cytokines interleukin (IL)-1β and IL-18. A variety of other downstream effects follows P2X7 activation over several minutes including shedding of certain surface molecules, membrane blebbing, microvesicle/exosome release and apoptosis of the cell. High concentrations of ATP (>100 µM) are required to activate P2X7 but it remains unclear where these levels exist, other than in inflammatory foci or confined spaces such as in bone. A variety of potent selective antagonists of P2X7 activation have recently become available, allowing clinical trials to be undertaken in inflammatory and immune-mediated disorders. Proteomic studies have shown that P2X7 exists as a large multiprotein complex which includes non-muscle myosin heavy chain and other elements of the cytoskeleton. In the absence of its ATP ligand and serum, P2X7 has an alternate function in the recognition and phagocytosis of non-opsonized foreign particles, including bacteria and apoptotic cells. The P2RX7 gene has many polymorphic variants and isoforms which increase or decrease function of the receptor. Genetic association studies have linked loss-of-function polymorphisms with reactivation of latent tuberculosis as well as symptomatic infection with certain other obligate intracellular pathogens. The many roles involving P2X7 suggest that this receptor is essential to fundamental aspects of the innate immune response.

Two haplotypes of the P2X7 receptor containing the Ala-348 to Thr polymorphism exhibit a gain-of-function effect and enhanced interleukin-1β secretion

The P2X(7) receptor is an ATP-gated cation channel expressed in immune cells and plays a role in proinflammatory cytokine release from monocytes and macrophages. This study investigated the coinheritance of 12 functionally relevant single nucleotide polymorphisms (SNPs) in the human P2X(7) gene (P2RX7), and the functional effect of each singly and in combination was assessed by measurements of ATP-induced currents and ethidium(+) uptake. Genotyping of 3430 Caucasian subjects identified 4 common haplotypes in addition to the common (wild-type) P2X(7)-1. Two haplotypes (denoted P2X(7)-2 and P2X(7)-4) contained various combinations of gain-of-function SNPs. P2X(7)-4 was identified uniquely by the Gln-460 to Arg polymorphism (rs2230912). When expressed in HEK-293 cells, recombinant P2X(7)-2, and P2X(7)-4 haplotypes displayed a 3-fold and 5-fold increase, respectively, in receptor function compared to the wild-type P2X(7)-1. Both P2X(7) haplotypes contained the Ala-348>Thr polymorphism (rs1718119), and this mutation was critical for the gain-of-function effect. Peripheral blood monocytes and erythrocytes from subjects homozygous for gain-of-function P2X(7) haplotypes exhibited increased ATP-induced ethidium(+) uptake and (86)Rb(+) efflux, respectively, and this correlated with increased IL-1beta secretion from LPS-primed monocytes. Inheritance of these P2X(7) haplotypes predisposing to increased proinflammatory cytokine secretion may be important in genetic association studies of inflammatory, infectious, and psychiatric disorders.

Genetics of the P2X7 receptor and human disease

The P2RX7 gene is highly polymorphic, and many single nucleotide polymorphisms (SNPs) underlie the wide variation observed in P2X7 receptor responses. We review the discovery of those non-synonymous SNPs that affect receptor function and compare their frequencies in different ethnic populations. Analysis of pairwise linkage disequilibrium (LD) predicts a limited range of haplotypes. The strong LD between certain functional SNPs provides insight into published studies of the association between SNPs and human disease.

The P2X7-nonmuscle myosin membrane complex regulates phagocytosis of nonopsonized particles and bacteria by a pathway attenuated by extracellular ATP

Phagocytosis of nonopsonized bacteria is central to innate immunity, but its regulation is less defined. We show that overexpression of the P2X(7) receptor greatly augments the phagocytosis of nonopsonized beads and heat-killed bacteria by transfected HEK-293 cells, whereas blocking P2X(7) expression by siRNA significantly reduces the phagocytic ability of human monocytic cells. An intact P2X(7)-nonmuscle myosin complex is required for phagocytosis of nonopsonized beads because activation of P2X(7) receptors by adenosine triphosphate (ATP), which dissociates myosin IIA from the P2X(7) complex, inhibits this phagocytic pathway. Fresh human monocytes rapidly phagocytosed live and heat-killed Staphylococcus aureus and Escherichia coli in the absence of serum, but the uptake was reduced by prior incubation with ATP, or P2X(7) monoclonal antibody, or recombinant P2X(7) extracellular domain. Injection of beads or bacteria into the peritoneal cavity of mice resulted in their brisk phagocytosis by macrophages, but injection of ATP before particles markedly decreased this uptake. These data demonstrate a novel pathway of phagocytosis of nonopsonized particles and bacteria, which operate in vivo and require an intact P2X(7)-nonmuscle myosin IIA membrane complex. The inhibitory effect of ATP on particle uptake by the macrophage is regulated by the P2X(7) receptor and defines this phagocytic pathway.

Point mutations confer loss of ATP-induced human P2X7 receptor function

Residues considered essential for ATP binding to the human P2X7 receptor (hP2X7R) were investigated. HEK293 cells or Xenopus oocytes were transfected with wild‐type or site‐directed mutants of hP2X7R 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 hP2X7R.

Extracellular ATP dissociates nonmuscle myosin from P2X7 complex: this dissociation regulates P2X7 pore formation

The P2X(7) receptor is a ligand-gated cation channel that is highly expressed on monocyte-macrophages and that mediates the pro-inflammatory effects of extracellular ATP. Dilation of the P2X(7) channel and massive K(+) efflux follows initial channel opening, but the mechanism of secondary pore formation is unclear. The proteins associated with P2X(7) were isolated by using anti-P2X(7) monoclonal antibody-coated Dynabeads from both interferon-gamma plus LPS-stimulated monocytic THP-1 cells and P2X(7)-transfected HEK-293 cells. Two nonmuscle myosins, NMMHC-IIA and myosin Va, were found to associate with P2X(7) in THP-1 cells and HEK-293 cells, respectively. Activation of the P2X(7) receptor by ATP caused dissociation of P2X(7) from nonmuscle myosin in both cell types. The interaction of P2X(7) and NMMHC-IIA molecules was confirmed by fluorescent life time measurements and fluorescent resonance of energy transfer-based time-resolved flow cytometry assay. Reducing the expression of NMMHC-IIA or myosin Va by small interfering RNA or short hairpin RNA led to a significant increase of P2X(7) pore function without any increase in surface expression or ion channel function of P2X(7) receptors. S-l-blebbistatin, a specific inhibitor of NMMHC-IIA ATPase, inhibited both ATP-induced ethidium uptake and ATP-induced dissociation of P2X(7)-NMMHC-IIA complex. In both cell types nonmuscle myosin closely interacts with P2X(7) and is dissociated from the complex by extracellular ATP. Dissociation of this anchoring protein may be required for the transition of P2X(7) channel to a pore.

A 5′ intronic splice site polymorphism leads to a null allele of the P2X7 gene in 1–2% of the Caucasian population

The P2X7 gene is important for the innate immune response but known polymorphisms do not explain all subjects with loss of P2X7 function. A splice site mutation (g → t) was found at position +1 of the first intron of the P2X7 gene in 7 of 336 Caucasians and 1 of 39 subjects of Indian ethnicity. All eight subjects were heterozygous for the uncommon 1513A → C polymorphism of the P2X7 gene. RT‐PCR and sequencing showed the splice site mutation was on the 1513C allele in the Caucasians and on the 1513A allele in the Indian subject. The splice site mutation is an inherited polymorphism and gives rise to a P2X7 null allele in 1–2% of the Caucasian population.

P2X 7 is a scavenger receptor for apoptotic cells in the absence of its ligand, extracellular ATP

Phagocytosis of apoptotic cells is essential during development and tissue remodeling. Our previous study has shown that the P2X7 receptor regulates phagocytosis of nonopsonized particles and bacteria. In this study, we demonstrate that P2X7 also mediates phagocytosis of apoptotic lymphocytes and neuronal cells by human monocyte-derived macrophages under serum-free conditions. ATP inhibited this process to a similar extent as observed with cytochalasin D. P2X7-transfected HEK-293 cells acquired the ability to phagocytose apoptotic lymphocytes. Injection of apoptotic thymocytes into the peritoneal cavity of wild-type mice resulted in their phagocytosis by macrophages, but injection of ATP prior to thymocytes markedly decreased this uptake. In contrast, ATP failed to inhibit phagocytosis of apoptotic thymocytes in vivo by P2X7-deficient peritoneal macrophages. The surface expression of P2X7 on phagocytes increased significantly during phagocytosis of either beads or apoptotic cells. A peptide screen library containing 24 biotin-conjugated peptides mimicking the extracellular domain of P2X7 was used to evaluate the binding profile to beads, bacteria, and apoptotic cells. One peptide showed binding to all particles and cell membrane lipids. Three other cysteine-containing peptides uniquely bound the surface of apoptotic cells but not viable cells, whereas substitution of alanine for cysteine abolished peptide binding. Several thiol-reactive compounds including N-acetyl-L-cysteine abolished phagocytosis of apoptotic SH-SY5Y cells by macrophages. These data suggest that the P2X7 receptor in its unactivated state acts like a scavenger receptor, and its extracellular disulphide bonds play an important role in direct recognition and engulfment of apoptotic cells.