Ross Corriden

ATP release guides neutrophil chemotaxis via P2Y2 and A3 receptors.

Cells must amplify external signals to orient and migrate in chemotactic gradient fields. We find that human neutrophils release adenosine triphosphate (ATP) from the leading edge of the cell surface to amplify chemotactic signals and direct cell orientation by feedback through P2Y2 nucleotide receptors. Neutrophils rapidly hydrolyze released ATP to adenosine that then acts via A3-type adenosine receptors, which are recruited to the leading edge, to promote cell migration. Thus, ATP release and autocrine feedback through P2Y2 and A3 receptors provide signal amplification, controlling gradient sensing and migration of neutrophils.

Autocrine regulation of T-cell activation by ATP release and P2X7 receptors

T‐cell activation requires the influx of extracellular calcium, although mechanistic details regarding such activation are not fully defined. Here, we show that P2X7 receptors play a key role in calcium influx and downstream signaling events associated with the activation of T cells. By real‐time PCR and immu‐nohistochemistry, we find that Jurkat T cells and human CD4+ T cells express abundant P2X7 receptors. We show, using a novel fluorescent microscopy technique, that T‐cell receptor (TCR) stimulation triggers the rapid release of ATP (<100 μM). This release of ATP is required for TCR‐mediated calcium influx, NFAT activation, and interleukin‐2 (IL‐2) production. TCR activation up‐regulates P2X7 receptor gene expression. Removal of extracellular ATP by apyrase or alkaline phosphatase treatment, inhibition of ATP release with the maxi‐anion channel blocker gadolinium chloride, or siRNA silencing of P2X7 receptors blocks calcium entry and inhibits T‐cell activation. Moreover, lymphocyte activation is impaired in C57BL/6 mice that express poorly functional P2X7 receptors, compared to control BALB/c mice, which express fully functional P2X7receptors. We conclude that ATP release and autocrine, positive feedback through P2X7 receptors is required for the effective activation of T cells.—Yip, L., Woe–hrle, T.,Corriden, R., Hirsh, M., Chen, Y., Inoue, Y., Ferrari, V., Insel, P.A., Junger, W.G. Autocrine regulation of T‐cell activation by ATP release and P2X7 receptors. FASEBJ. 23, 1685–1693 (2009)

Ecto-nucleoside Triphosphate Diphosphohydrolase 1 (E-NTPDase1/CD39) Regulates Neutrophil Chemotaxis by Hydrolyzing Released ATP to Adenosine

Polymorphonuclear neutrophils release ATP in response to stimulation by chemoattractants, such as the peptide N-formyl-methionyl-leucyl-phenylalanine. Released ATP and the hydrolytic product adenosine regulate chemotaxis of neutrophils by sequentially activating purinergic nucleotide and adenosine receptors, respectively. Here we show that that ecto-nucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1, CD39) is a critical enzyme for hydrolysis of released ATP by neutrophils and for cell migration in response to multiple agonists (N-formyl-methionyl-leucyl-phenylalanine, interleukin-8, and C5a). Upon stimulation of human neutrophils or differentiated HL-60 cells in a chemotactic gradient, E-NTPDase1 tightly associates with the leading edge of polarized cells during chemotaxis. Inhibition of E-NTPDase1 reduces the migration speed of neutrophils but not their ability to detect the orientation of the gradient field. Studies of neutrophils from E-NTPDase1 knock-out mice reveal similar impairments of chemotaxis in vitro and in vivo. Thus, E-NTPDase1 plays an important role in regulating neutrophil chemotaxis by facilitating the hydrolysis of extracellular ATP.

New insights regarding the regulation of chemotaxis by nucleotides, adenosine, and their receptors

The directional movement of cells can be regulated by ATP, certain other nucleotides (e.g., ADP, UTP), and adenosine. Such regulation occurs for cells that are “professional phagocytes” (e.g., neutrophils, macrophages, certain lymphocytes, and microglia) and that undergo directional migration and subsequent phagocytosis. Numerous other cell types (e.g., fibroblasts, endothelial cells, neurons, and keratinocytes) also change motility and migration in response to ATP, other nucleotides, and adenosine. In this article, we review how nucleotides and adenosine modulate chemotaxis and motility and highlight the importance of nucleotide- and adenosine-regulated cell migration in several cell types: neutrophils, microglia, endothelial cells, and cancer cells. We also discuss difficulties in conducting experiments and drawing conclusions regarding the ability of nucleotides and adenosine to modulate the migration of professional and non-professional phagocytes.

Azithromycin Synergizes with Cationic Antimicrobial Peptides to Exert Bactericidal and Therapeutic Activity Against Highly Multidrug-Resistant Gram-Negative Bacterial Pathogens

Antibiotic resistance poses an increasingly grave threat to the public health. Of pressing concern, rapid spread of carbapenem-resistance among multidrug-resistant (MDR) Gram-negative rods (GNR) is associated with few treatment options and high mortality rates. Current antibiotic susceptibility testing guiding patient management is performed in a standardized manner, identifying minimum inhibitory concentrations (MIC) in bacteriologic media, but ignoring host immune factors. Lacking activity in standard MIC testing, azithromycin (AZM), the most commonly prescribed antibiotic in the U.S., is never recommended for MDR GNR infection. Here we report a potent bactericidal action of AZM against MDR carbapenem-resistant isolates of Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii. This pharmaceutical activity is associated with enhanced AZM cell penetration in eukaryotic tissue culture media and striking multi-log-fold synergies with host cathelicidin antimicrobial peptide LL-37 or the last line antibiotic colistin. Finally, AZM monotherapy exerts clear therapeutic effects in murine models of MDR GNR infection. Our results suggest that AZM, currently ignored as a treatment option, could benefit patients with MDR GNR infections, especially in combination with colistin.

Tamoxifen augments the innate immune function of neutrophils through modulation of intracellular ceramide

Tamoxifen is a selective estrogen receptor modulator widely used for the treatment of breast cancer. In addition to its activity as an estrogen receptor agonist/antagonist, tamoxifen also modulates sphingolipid biosynthesis, which has been shown to play an important role in the regulation of neutrophil activity. Here, we find that tamoxifen stimulation enhances several pro-inflammatory pathways in human neutrophils, including chemotaxis, phagocytosis and neutrophil extracellular trap (NET) formation. The enhancement of NET production occurs via a ceramide/PKCζ-mediated pathway, and treatment with synthetic ceramide is sufficient to promote NET formation. Pretreatment of human neutrophils with tamoxifen boosts neutrophil bactericidal capacity against a variety of pathogens in vitro and enhances clearance of the leading human pathogen methicillin-resistant Staphylococcus aureus in vivo. Our results suggest that tamoxifen, and the lipid signaling pathways it modulates, merit further exploration as targets for boosting host innate immune function.

Hypertonic stress regulates T-cell function by the opposing actions of extracellular adenosine triphosphate and adenosine.

Hypertonic saline (HS) treatment promotes interleukin (IL)-2 production and enhances T-cell activation by the release of cellular adenosine triphosphate (ATP) that activates P2 nucleotide receptors. Released ATP can be hydrolyzed to adenosine, which inhibits T-cell activation. We examined if adenosine affects the response of T cells to HS treatment, and found that the amount of ATP released from T cells is a function of the HS concentration and duration of HS exposure. Physiologically relevant HS concentrations (<40 mmol/L) induced rapid ATP release, with the highest ATP concentrations released within 1 min. The released ATP was converted to adenosine, which opposed the enhancing effects of HS on IL-2 production. We found that Jurkat and CD4+ primary human T cells express most abundantly the A2A and A2B adenosine receptor subtypes, which mediate the suppressive effects of adenosine, as the A2 receptor agonist CGS 21680 suppressed IL-2 production, whereas the A2 receptor antagonist 3,7-dimethyl-1-(2-propynyl)xanthine augmented the enhancing effect of HS on T-cell function. Elimination of extracellular adenosine by adding exogenous adenosine deaminase also increased the enhancing effects of HS. These data suggest that the effect of HS treatment on T-cell function can be modulated with pharmacological agents that abolish the suppressive effects of adenosine formed from the ATP that is released in response to HS treatment.ABBREVIATIONS-(HS) hypertonic saline, (ADA) adenosine deaminase, (CPA) N6-cyclopentyladenosine, (CGS 21680) 2-p-(2-carboxyethyl)phenethylamino-5&vprime;-N-ethylcarboxamidoadenosine hydrochloride, (IB-MECA) 1-deoxy-1-[6-[((3-iodophenyl)methyl)amino]-9H-purin-9-yl]-N-methyl-&bgr;-D-ribofuranuronamide N6-(3-iodobenzyl)adenosine-5&vprime;-N-methyluronamide, (EHNA) erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride, (DIPY) dipyridamole, (SCH 58261) 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine, (DMPX) 3,7-dimethyl-1-(2-propynyl)xanthine, (8-SPT) 8-(p-sulfophenyl) theophylline hydrate, (&Dgr;&Dgr;Ct) comparative threshold cycle

Kinetic analysis of antagonist-occupied adenosine-A3 receptors within membrane microdomains of individual cells provides evidence of receptor dimerization and allosterism

In our previous work, using a fluorescent adenosine‐A3 receptor (A3AR) agonist and fluorescence correlation spectroscopy (FCS), we demonstrated high‐affinity labeling of the active receptor (R∗) conformation. In the current study, we used a fluorescent A3AR antagonist (CA200645) to study the binding characteristics of antagonist‐occupied inactive receptor (R) conformations in membrane microdomains of individual cells. FCS analysis of CA200645‐occupied A3ARs revealed 2 species, τD2 and τD3, that diffused at 2.29 ± 0.35 and 0.09 ± 0.03 μm2/s, respectively. FCS analysis of a green fluorescent protein (GFP)‐tagged A3AR exhibited a single diffusing species (0.105 μm2 /s). The binding of CA200645 to τD3 was antagonized by nanomolar concentrations of the A3 antagonist MRS 1220, but not by the agonist NECA (up to 300 nM), consistent with labeling of R. CA200645 normally dissociated slowly from the A3AR, but inclusion of xanthine amine congener (XAC) or VUF 5455 during washout markedly accelerated the reduction in the number of particles exhibiting τD3 characteristics. It is notable that this effect was accompanied by a significant increase in the number of particles with τD2 diffusion. These data show that FCS analysis of ligand‐occupied receptors provides a unique means of monitoring ligand A3AR residence times that are significantly reduced as a consequence of allosteric interaction across the dimer interface.—Corriden, R., Kilpatrick, L. E., Kellam, B., Briddon, S. J., Hill, S. J., Kinetic analysis of antagonist‐occupied adenosine‐A3 receptors within membrane microdomains of individual cells provides evidence for receptor dimerization and allosterism. FASEB J. 28, 4211‐4222 (2014). www.fasebj.org

Skin-Resident T Cells Sense Ultraviolet Radiation–Induced Injury and Contribute to DNA Repair

Skin-resident T cells have been shown to play important roles in tissue homeostasis and wound repair, but their role in UV radiation (UVR)–mediated skin injury and subsequent tissue regeneration is less clear. In this study, we demonstrate that acute UVR rapidly activates skin-resident T cells in humans and dendritic epidermal γδ T cells (DETCs) in mice through mechanisms involving the release of ATP from keratinocytes. Following UVR, extracellular ATP leads to an increase in CD69 expression, proliferation, and IL-17 production, and to changes in DETC morphology. Furthermore, we find that the purinergic receptor P2X7 and caspase-1 are necessary for UVR-induced IL-1 production in keratinocytes, which increases IL-17 secretion by DETCs. IL-17, in turn, induces epidermal TNF-related weak inducer of apoptosis and growth arrest and DNA damage–associated gene 45, two molecules linked to the DNA repair response. Finally, we demonstrate that DETCs and human skin-resident T cells limit DNA damage in keratinocytes. Taken together, our findings establish a novel role for skin-resident T cells in the UVR-associated DNA repair response and underscore the importance of skin-resident T cells to overall skin regeneration.

Adenosine-A3 receptors in neutrophil microdomains promote the formation of bacteria-tethering cytonemes.

The A3‐adenosine receptor (A3AR) has recently emerged as a key regulator of neutrophil behaviour. Using a fluorescent A3AR ligand, we show that A3ARs aggregate in highly polarized immunomodulatory microdomains on human neutrophil membranes. In addition to regulating chemotaxis, A3ARs promote the formation of filipodia‐like projections (cytonemes) that can extend up to 100 μm to tether and ‘reel in’ pathogens. Exposure to bacteria or an A3AR agonist stimulates the formation of these projections and bacterial phagocytosis, whereas an A3AR‐selective antagonist inhibits cytoneme formation. Our results shed new light on the behaviour of neutrophils and identify the A3AR as a potential target for modulating their function.