Sébastien A. Lévesque

Comparative hydrolysis of P2 receptor agonists by NTPDases 1, 2, 3 and 8

Nucleoside triphosphate diphosphohydrolases 1, 2, 3 and 8 (NTPDases 1, 2, 3 and 8) are the dominant ectonucleotidases and thereby expected to play important roles in nucleotide signaling. Distinct biochemical characteristics of individual NTPDases should allow them to regulate P2 receptor activation differentially. Therefore, the biochemical and kinetic properties of these enzymes were compared. NTPDases 1, 2, 3 and 8 efficiently hydrolyzed ATP and UTP with Km values in the micromolar range, indicating that they should terminate the effects exerted by these nucleotide agonists at P2X1- and P2Y2,4,11 receptors. Since NTPDase1 does not allow accumulation of ADP, it should terminate the activation of P2Y1,12,13 receptors far more efficiently than the other NTPDases. In contrast, NTPDases 2, 3 and 8 are expected to promote the activation of ADP specific receptors, because in the presence of ATP they produce a sustained (NTPDase2) or transient (NTPDases 3 and 8) accumulation of ADP. Interestingly, all plasma membrane NTPDases dephosphorylate UTP with a significant accumulation of UDP, favoring P2Y6 receptor activation. NTPDases differ in divalent cation and pH dependence, although all are active in the pH range of 7.0-.5. Various NTPDases may also distinctly affect formation of extracellular adenosine and therefore adenosine receptor-mediated responses, since they generate different amounts of the substrate (AMP) and inhibitor (ADP) of ecto-5-nucleotidase, the rate limiting enzyme in the production of adenosine. Taken together, these data indicate that plasma membrane NTPDases hydrolyze nucleotides in a distinctive manner and may therefore differentially regulate P2 and adenosine receptor signaling.

Specificity of the ecto-ATPase inhibitor ARL 67156 on human and mouse ectonucleotidases

ARL 67156, 6‐N,N‐Diethyl‐D‐β‐γ‐dibromomethylene adenosine triphosphate, originally named FPL 67156, is the only commercially available inhibitor of ecto‐ATPases. Since the first report on this molecule, various ectonucleotidases responsible for the hydrolysis of ATP at the cell surface have been cloned and characterized. In this work, we identified the ectonucleotidases inhibited by ARL 67156.

Impact of Ectoenzymes on P2 and P1 Receptor Signaling

P2 receptors that are activated by extracellular nucleotides (e.g., ATP, ADP, UTP, UDP, Ap(n)A) and P1 receptors activated by adenosine control a diversity of biological processes. The activation of these receptors is tightly regulated by ectoenzymes that metabolize their ligands. This review presents these enzymes as well as their roles in the regulation of P2 and P1 receptor activation. We focus specifically on the role of ectoenzymes in processes of our interest, that is, inflammation, vascular tone, and neurotransmission. An update on the development of ectonucleotidase inhibitors is also presented.

NTPDase1 governs P2X7-dependent functions in murine macrophages.

P2X7 receptor is an adenosine triphosphate (ATP)‐gated ion channel within the multiprotein inflammasome complex. Until now, little is known about regulation of P2X7 effector functions in macrophages. In this study, we show that nucleoside triphosphate diphosphohydrolase 1 (NTPDase1)/CD39 is the dominant ectonucleotidase expressed by murine peritoneal macrophages and that it regulates P2X7‐dependent responses in these cells. Macrophages isolated from NTPDase1‐null mice (Entpd1−/−) were devoid of all ADPase and most ATPase activities when compared with WT macrophages (Entpd1+/+). Entpd1−/− macrophages exposed to millimolar concentrations of ATP were more susceptible to cell death, released more IL‐1β and IL‐18 after TLR2 or TLR4 priming, and incorporated the fluorescent dye Yo‐Pro‐1 more efficiently (suggestive of increased pore formation) than Entpd1+/+ cells. Consistent with these observations, NTPDase1 regulated P2X7‐associated IL‐1β release after synthesis, and this process occurred independently of, and prior to, cytokine maturation by caspase‐1. NTPDase1 also inhibited IL‐1β release in vivo in the air pouch inflammatory model. Exudates of LPS‐injected Entpd1−/− mice had significantly higher IL‐1β levels when compared with Entpd1+/+ mice. Altogether, our studies suggest that NTPDase1/CD39 plays a key role in the control of P2X7‐dependent macrophage responses.

Neutrophils Mediate Blood–Spinal Cord Barrier Disruption in Demyelinating Neuroinflammatory Diseases

Disruption of the blood–brain and blood–spinal cord barriers (BBB and BSCB, respectively) and immune cell infiltration are early pathophysiological hallmarks of multiple sclerosis (MS), its animal model experimental autoimmune encephalomyelitis (EAE), and neuromyelitis optica (NMO). However, their contribution to disease initiation and development remains unclear. In this study, we induced EAE in lys-eGFP-ki mice and performed single, nonterminal intravital imaging to investigate BSCB permeability simultaneously with the kinetics of GFP+ myeloid cell infiltration. We observed a loss in BSCB integrity within a day of disease onset, which paralleled the infiltration of GFP+ cells into the CNS and lasted for ∼4 d. Neutrophils accounted for a significant proportion of the circulating and CNS-infiltrating myeloid cells during the preclinical phase of EAE, and their depletion delayed the onset and reduced the severity of EAE while maintaining BSCB integrity. We also show that neutrophils collected from the blood or bone marrow of EAE mice transmigrate more efficiently than do neutrophils of naive animals in a BBB cell culture model. Moreover, using intravital videomicroscopy, we demonstrate that the IL-1R type 1 governs the firm adhesion of neutrophils to the inflamed spinal cord vasculature. Finally, immunostaining of postmortem CNS material obtained from an acutely ill multiple sclerosis patient and two neuromyelitis optica patients revealed instances of infiltrated neutrophils associated with regions of BBB or BSCB leakage. Taken together, our data provide evidence that neutrophils are involved in the initial events that take place during EAE and that they are intimately linked with the status of the BBB/BSCB.

Implication of an AGT haplotype in a multigene association study with pregnancy hypertension.

Abstract—Several association studies of candidate genes for preeclampsia and essential hypertension have led to discordant results, partly because of small sample sizes. Using a large population-based sample of pregnant women, we conducted an association study of 10 polymorphisms in 9 genes and aimed (1) to validate 10 published associations with preeclampsia or essential hypertension, (2) to investigate candidate polymorphisms previously associated with preeclampsia for association with essential hypertension and similarly with polymorphisms previously associated with essential hypertension. From a prospective sample of 3391 nulliparous French Canadian pregnant women, we identified 180 cases of preeclampsia, 203 cases of essential hypertension that were matched with normotensive control subjects (n=310 and 357, respectively). Polymorphisms were genotyped by allele-specific PCR. Among our candidate polymorphisms, the Met allele of Thr174Met of AGT was associated with preeclampsia (P =0.0033). Haplotype analysis revealed that the A-Met-Thr (G1035A-Thr174Met-Met235Thr) haplotype was associated with a 2.1-fold increased risk of preeclampsia (95% CI, 1.4 to 3.4; P =0.0008). In conclusion, we observed a strong association between a specific AGT haplotype and preeclampsia in our population, without replicating previous published associations with either preeclampsia or essential hypertension. Our data support a role for AGT in genetic susceptibility to preeclampsia.

Extracellular ATP and P2 receptors are required for IL-8 to induce neutrophil migration.

The chemokine interleukin 8 (IL-8) is a major chemoattractant for human neutrophils. Here, we demonstrate novel evidence that IL-8-induced neutrophil chemotaxis requires a concurrent activation of P2 receptors, most likely the P2Y(2) which is dominantly expressed in these cells. Indeed, the migration of human neutrophils towards IL-8 was significantly inhibited by the P2Y receptor antagonists, suramin and reactive blue 2 (RB-2) and potentiated by a P2Y(2) ligand, ATP, but insensitive to specific antagonists of P2Y(1), P2Y(6) and P2Y(11) receptors. Adenosine had no effect on neutrophil migration towards IL-8 which contrasted with the stimulatory effect of this molecule on neutrophil chemotaxis caused by formyl-Met-Leu-Phe (fMLP or fMLF). Taken together, these data suggest that extracellular ATP is necessary for IL-8 to exert its chemotactic effect on neutrophils.

A highly sensitive CE‐UV method with dynamic coating of silica‐fused capillaries for monitoring of nucleotide pyrophosphatase/phosphodiesterase reactions

A new highly sensitive capillary electrophoresis (CE) method applying dynamic coating and on‐line stacking for the monitoring of nucleotide pyrophosphatases/phosphodiesterases (NPPs) and the screening of inhibitors was developed. NPP1 and NPP3 are membrane glycoproteins that catalyze the hydrolysis nucleotides, e.g. convert adenosine 5′‐triphosphate to adenosine 5′‐monophosphate (AMP) and pyrophosphate. Enzymatic reactions were performed and directly subjected to CE analysis. Since the enzymatic activity was low, standard methods were insufficient. The detection of nanomolar AMP and other nucleotides could be achieved by field‐enhanced sample injection and the addition of polybrene to the running buffer. The polycationic polymer caused a dynamic coating of the silica‐fused capillary, resulting in a reversed electroosmotic flow. The nucleotides migrated in the direction of the electroosmotic flow, whereas the positively charged polybrene molecules moved in the opposite direction, resulting in a narrow sample zone over a long injection time. Using this on‐line sensitivity enhancement technique, a more than 70‐fold enrichment was achieved for AMP (limit of detection, 46 nM) along with a short migration time (5 min) without compromising separation efficiency and peak shape. The optimized CE conditions were as follows: fused‐silica capillary (30 cm effective length×75 μm), electrokinetic injection for 60 s, 50 mM phosphate buffer pH 6.5, 0.002% polybrene, constant current of −60 μA, UV detection at 210 nm, uridine 5′‐monophosphate as the internal standard. The new method was used to study enzyme kinetics and inhibitors. It opens an easy way to determine the activities of slowly metabolizing enzymes such as NPPs, which are of considerable interest as novel drug targets.

Central canal ependymal cells proliferate extensively in response to traumatic spinal cord injury but not demyelinating lesions.

The adult mammalian spinal cord has limited regenerative capacity in settings such as spinal cord injury (SCI) and multiple sclerosis (MS). Recent studies have revealed that ependymal cells lining the central canal possess latent neural stem cell potential, undergoing proliferation and multi-lineage differentiation following experimental SCI. To determine whether reactive ependymal cells are a realistic endogenous cell population to target in order to promote spinal cord repair, we assessed the spatiotemporal dynamics of ependymal cell proliferation for up to 35 days in three models of spinal pathologies: contusion SCI using the Infinite Horizon impactor, focal demyelination by intraspinal injection of lysophosphatidylcholine (LPC), and autoimmune-mediated multi-focal demyelination using the active experimental autoimmune encephalomyelitis (EAE) model of MS. Contusion SCI at the T9–10 thoracic level stimulated a robust, long-lasting and long-distance wave of ependymal proliferation that peaked at 3 days in the lesion segment, 14 days in the rostral segment, and was still detectable at the cervical level, where it peaked at 21 days. This proliferative wave was suppressed distal to the contusion. Unlike SCI, neither chemical- nor autoimmune-mediated demyelination triggered ependymal cell proliferation at any time point, despite the occurrence of demyelination (LPC and EAE), remyelination (LPC) and significant locomotor defects (EAE). Thus, traumatic SCI induces widespread and enduring activation of reactive ependymal cells, identifying them as a robust cell population to target for therapeutic manipulation after contusion; conversely, neither demyelination, remyelination nor autoimmunity appears sufficient to trigger proliferation of quiescent ependymal cells in models of MS-like demyelinating diseases.

NTPDase1 Controls IL-8 Production by Human Neutrophils

The ectonucleotidase NTPDase1 (CD39) terminates P2 receptor activation by the hydrolysis of extracellular nucleotides (i.e., the P2 receptor ligands). In agreement with that role, exacerbated inflammation has been observed in NTPDase1-deficient mice. In this study, we extend these observations by showing that inhibition of NTPDase1 markedly increases IL-8 production by TLR-stimulated human neutrophils. First, immunolabeling of human blood neutrophils and neutrophil-like HL60 cells displayed the expression of NTPDase1 protein, which correlated with the hydrolysis of ATP at their surface. NTPDase1 inhibitors (e.g., NF279 and ARL 67156) as well as NTPDase1-specific small interfering RNAs markedly increased IL-8 production in neutrophils stimulated with LPS and Pam3CSK4 (agonists of TLR4 and TLR1/2, respectively) but not with flagellin (TLR5) and gardiquimod (TLR7 and 8). This increase in IL-8 release was due to the synergy between TLRs and P2 receptors. Indeed, ATP was released from neutrophils constitutively and accumulated in the medium upon NTPDase1 inhibition by NF279. Likewise, both human blood neutrophils and neutrophil-like HL60 cells produced IL-8 in response to exogenous nucleotides, ATP being the most potent inducer. In agreement, P2Y2 receptor knockdown in neutrophil-like HL60 cells markedly decreased LPS- and Pam3CSK4-induced IL-8 production. In line with these in vitro results, injection of LPS in the air pouches of NTPDase1-deficient mice triggered an increased production of the chemokines MIP-2 and keratinocyte-derived chemokine (i.e., the rodent counterparts of human IL-8) compared with that in wild-type mice. In summary, NTPDase1 controls IL-8 production by human neutrophils via the regulation of P2Y2 activation.