James S. Wiley

CD4 Effector T Cell Subsets in the Response to Influenza Heterogeneity, Migration, and Function

The immune response of naive CD4 T cells to influenza virus is initiated in the draining lymph nodes and spleen, and only after effectors are generated do antigen-specific cells migrate to the lung which is the site of infection. The effector cells generated in secondary organs appear as multiple subsets which are a heterogeneous continuum of cells in terms of number of cell divisions, phenotype and function. The effector cells that migrate to the lung constitute the more differentiated of the total responding population, characterized by many cell divisions, loss of CD62L, down-regulation of CCR7, stable expression of CD44 and CD49d, and transient expression of CCR5 and CD25. These cells also secrete high levels of interferon γ and reduced levels of interleukin 2 relative to those in the secondary lymphoid organs. The response declines rapidly in parallel with viral clearance, but a spectrum of resting cell subsets reflecting the pattern at the peak of response is retained, suggesting that heterogeneous effector populations may give rise to corresponding memory populations. These results reveal a complex response, not an all-or-none one, which results in multiple effector phenotypes and implies that effector cells and the memory cells derived from them can display a broad spectrum of functional potentials.

Genome-wide meta-analysis identifies novel multiple sclerosis susceptibility loci

To perform a 1‐stage meta‐analysis of genome‐wide association studies (GWAS) of multiple sclerosis (MS) susceptibility and to explore functional consequences of new susceptibility loci.

The isoquinoline derivative KN-62 a potent antagonist of the P2Z-receptor of human lymphocytes.

Extracellular adenosine 5′‐triphosphate (ATP) is an agonist for a P2Z receptor on human lymphocytes which mediates opening of a cation‐selective ion channel, activation of phospholipase D and shedding of the adhesion molecule, L‐selectin, from the cell surface. The isoquinolinesulphonamides, KN‐62, (1‐[N, O‐bis(5‐isoquinolinesulphonyl)‐N‐methyl‐L‐tyrosyl]‐ 4‐phenylpiperazine), a selective antagonist of Ca2+/calmodulin‐dependent protein kinase II (CaMKII), and KN‐04, (N‐[1‐[N‐methyl‐p‐(5 isoquinoline sulphonyl)benzyl]‐2‐(4 phenylpiperazine)ethyl]‐5‐isoquinolinesulphonamide) an inactive analogue, were used to investigate the possible role of CaMKII in these diverse effects of extracellular ATP. KN‐62 potently antagonized ATP‐stimulated Ba2+ influx into fura‐2 loaded human lymphocytes with an IC50 of 12.7±1.5 nm (n=3) and complete inhibition of the flux at a concentration of 500 nm. Similarly, KN‐62 inhibited ATP‐stimulated ethidium+ uptake, measured by time resolved flow cytometry, with an IC50 of 13.1±2.6 nm (n=4) and complete inhibition of the flux at 500 nm. KN‐04 antagonized ATP‐stimulated Ba2+ influx with an IC50 of 17.3±2.7 nm (n=3). Similarly, KN‐04 inhibited ATP‐stimulated ethidium+ uptake with an IC50 of 37.2±8.9 nm (n=4). Both fluxes were completely inhibited at 500 nm KN‐04. ATP‐stimulated phospholipase D activity, measured in [3H]‐oleic acid‐labelled lymphocytes by the transphosphatidylation reaction, was antagonized by KN‐62 and KN‐04, with 50% inhibition at 5.9±1.2 and 9.7±2.8 nm (n=3), respectively. Both KN‐62 and KN‐04 inhibited ATP‐stimulated shedding of L‐selectin, measured by flow cytometric analysis of cell surface L‐selectin, with IC50 values of 31.5±4.5 and 78.7±10.8 nm (n=3), respectively. Neither of the isoquinolinesulphonamides (500 nm) inhibited phorbol ester‐ or ionomycin‐stimulated phospholipase D activity or phorbol ester‐induced shedding of L‐selectin. The inhibitory effect of KN‐62 or KN‐04 on P2Z‐mediated responses was slow in onset (5 min) and only partially reversed by washing the cells. Both KN‐62 and KN‐04 (at 500 nm) had no effect on uridine 5′‐triphosphate (UTP)‐stimulated Ca2+ transients in fura‐2 loaded human neutrophils, a response which is mediated by the P2Y2 receptor. Thus, KN‐62 and KN‐04 are potent antagonists of the P2Z receptor and at nanomolar concentrations inhibit all known responses mediated by the P2Z receptor of human lymphocytes. In contrast, KN‐62 and KN‐04 had no effect on responses mediated by the P2Y2 receptor of neutrophils. Moreover, since KN‐62 and KN‐04 are almost equipotent, the P2Z‐mediated responses do not involve CaMKII, but indicate that the isoquinolinesulphonamides are potent and direct inhibitors of the P2Z‐receptor.

Filamin A mutations cause periventricular heterotopia with Ehlers-Danlos syndrome

Objective: To define the clinical, radiologic, and genetic features of periventricular heterotopia (PH) with Ehlers-Danlos syndrome (EDS). Methods: Exonic sequencing and single stranded conformational polymorphism (SSCP) analysis was performed on affected individuals. Linkage analysis using microsatellite markers on the X-chromosome was performed on a single pedigree. Western blotting evaluated for loss of filamin A (FLNA) protein and Southern blotting assessed for any potential chromosome rearrangement in this region. Results: The authors report two familial cases and nine additional sporadic cases of the EDS-variant form of PH, which is characterized by nodular brain heterotopia, joint hypermobility, and development of aortic dilatation in early adulthood. MRI typically demonstrated bilateral nodular PH, indistinguishable from PH due to FLNA mutations. Exonic sequencing or SSCP analyses of FLNA revealed a 2762 delG single base pair deletion in one affected female. Another affected female harbored a C116 single point mutation, resulting in an A39G change. A third affected female had a 4147 delG single base pair deletion. One pedigree with no detectable exonic mutation demonstrated positive linkage to the FLNA locus Xq28, an affected individual in this family also had no detectable FLNA protein, but no chromosomal rearrangement was detected. Conclusion: These results suggest that the Ehlers-Danlos variant of periventricular heterotopia (PH), in part, represents an overlapping syndrome with X-linked dominant PH due to filamin A mutations.

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.

Glu496 to Ala Polymorphism in the P2X7 Receptor Impairs ATP-Induced IL-1β Release from Human Monocytes

Priming of monocytes with LPS produces large quantities of intracellular, biologically inactive IL-1β that can be processed and released by subsequent activation of the P2X7 receptor by extracellular ATP. We examined whether a loss-of-function polymorphism of the human P2X7 receptor (Glu496Ala) impairs this process. Both ATP-induced ethidium+ uptake and ATP-induced shedding of L-selectin (CD62L) were nearly absent in monocytes from four subjects homozygous for Glu496Ala confirming that this polymorphism impairs P2X7 function. The level of ATP-induced IL-1β released in 2 h from LPS-activated whole blood from homozygous subjects was 50% of that from wild-type samples. A more marked defect in IL-1β release was observed from LPS-activated monocytes of homozygous subjects which was only 22% of that released from wild-type monocytes after a 30-min incubation with ATP. However, after a 60-min incubation with ATP, the amount of IL-1β released from homozygous monocytes was 70% of that released from wild-type monocytes. Incubation of monocytes of either genotype with nigericin resulted in a similar release of IL-1β. Western blotting demonstrated that ATP induced the release of mature 17-kDa IL-1β from monocytes, and confirmed that this process was impaired in homozygous monocytes. Finally, ATP-induced 86Rb+ efflux was 9-fold lower from homozygous monocytes than from wild-type monocytes. The results indicate that ATP-induced release of IL-1β is slower in monocytes from subjects homozygous for the Glu496Ala polymorphism in the P2X7 receptor and that this reduced rate of IL-1β release is associated with a lower ATP-induced K+ efflux.

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.

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

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 (1513A→C), with 1–2% prevalence in the homozygous state, which codes for a nonfunctional receptor. IFN-γ-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.

Extracellular ATP causes loss of L-selectin from human lymphocytes via occupancy of P2Z purinoceptors

Lymphocytes from normal subjects or patients with chronic lymphocytic leukemia are known to possess receptors for extracellular ATP termed P2Z purinoceptors whose physiological role is undefined. Addition of extracellular ATP (50–500 μM) to both normal and leukemic lymphocytes caused loss of binding of monoclonal antibodies to L‐selectin (CD62L) on the cell surface. UTP, ADP, and adenosine (all at 500 μM) had no effect on L‐selectin expression. Several features of the ATP‐induced loss of L selectin indicate that this effect is mediated by lymphocyte P2Z purinoceptors. First the loss was attenuated in isotonic NaCl medium compared to 150 mM KCl medium. Second the loss of L‐selectin was immediately halted by addition of Mg2+ ions in molar excess of ATP. The most potent nucleotide causing L‐selectin loss was benzoylbenzoic ATP (>10 μM) which is also the most potent agonist for the P2Z purinoceptor. Finally preincubation of lymphocytes with oxidized ATP, an irreversible inhibitor of P2Z purinoceptors, also inhibited ATP induced loss of L‐selectin. Extracellular ATP is known to open an ion channel associated with the P2Z purinoceptor on B‐lymphocytes which allows influx of Ca2+. However, ATP‐induced loss of L‐selectin did not require extracellular Ca2+. Moreover addition of the calcium ionophore, ionomycin, had minimal effect on L‐selectin expression. Staurosporine (500 nM), an inhibitor of protein kinase C, inhibited only 10% of ATP induced loss of L‐selectin but completely inhibited the loss of L‐selectin caused by 50 nM PMA. Thus extracellular ATP interacts with lymphocyte P2Z purinoceptors which leads to shedding of L‐selectin via a pathway which requires neither Ca2+ influx nor activation of protein kinase C. ATP may have a physiological role in the loss of L‐selectin which occurs during the interactions of lymphocytes with other cells.