Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.

Subscribe

Logo for

Science 314 (5806): 1792-1795

Copyright © 2006 by the American Association for the Advancement of Science

ATP Release Guides Neutrophil Chemotaxis via P2Y2 and A3 Receptors

Yu Chen1*, Ross Corriden1,2*, Yoshiaki Inoue1, Linda Yip1, Naoyuki Hashiguchi1, Annelies Zinkernagel4, Victor Nizet4, Paul A. Insel2,3, and Wolfgang G. Junger1{dagger}

1 Department of Surgery, University of California San Diego, San Diego, CA 92103, USA.
2 Department of Pharmacology, University of California San Diego, La Jolla, CA 92093, USA.
3 Department of Medicine, University of California San Diego, La Jolla, CA 92093, USA.
4 Department of Pediatrics, University of California San Diego, La Jolla, CA 92093, USA.


Figure 1 Fig. 1.. Release of ATP by FMLP-stimulated neutrophils and formation of adenosine. (A) ATP released in response to FMLP was determined with high-performance liquid chromatography (HPLC) analysis of extracellular ATP and its breakdown products. A pair of chromatograms shows ATP and its hydrolytic products in the extracellullar environment of neutrophils before (lower trace) or after (upper trace) stimulation with 100 nM FMLP. ADP, adenosine diphosphate. (B) Extracellular concentrations of ATP, AMP, and adenosine measured with HPLC analysis at different time points after stimulation of neutrophils with 100 nM FMLP. (C) Extracellular ATP, AMP, and adenosine at different times after addition of exogenous ATP to isolated neutrophils. Error bars indicate SD of triplicate determinations. (D) Release of ATP from living cells stimulated in a gradient field of FMLP generated with a micropipette (asterisks) was visualized with the use of a two-enzyme assay system that catalyzes the conversion of NADP+ to NADPH in the presence of ATP. The generation of NADPH was monitored in real time by means of fluorescence microscopy (excitation wavelength, 340 nm; emission wavelength, 460 nm). Images were captured at 12 frames per minute. (Inset) Extracellular ATP concentrations based on calibration of gray values with a known ATP standard. [View Larger Version of this Image (39K GIF file)]
 

Figure 2 Fig. 2.. Effect of exogenous ATP on neutrophil chemotaxis. Trans-well assays with neutrophils in upper wells separated from lower wells containing 1 nM FMLP by a filter with 3-µm pore size were used to assess chemotaxis. (A) Effect of apyrase added to the lower or upper well on neutrophil chemotaxis and on FMLP-induced oxidative burst. Cell responses are expressed as a percent of the response to FMLP in the absence of apyrase. (B) Treatment with ATP-{gamma}-S in the presence (circles) or absence (diamonds) of FMLP. (C) Cell migration studied under the microscope was analyzed by tracing the paths of cells migrating toward a micropipette tip containing 100 nM FMLP in the absence (left) or presence (middle) of 10 U/ml of apyrase or 100 µM ATP-{gamma}-S (right). The y axis of the traces represents the direction toward the chemoattractant source, and the x axis shows the deviation from the straight path. Cell traces were arranged to show their origins at x = y =0.(D) Effect of the P2-receptor antagonist suramin on FMLP-induced cell migration. Error bars in (A), (B), and (D) indicate SD of triplicate determinations. [View Larger Version of this Image (39K GIF file)]
 

Figure 3 Fig. 3.. P1- and P2-receptor expression in neutrophils (A) and HL60 cells (B). P1- and P2-receptor mRNA expression in human neutrophils and HL60 cells was estimated with real-time RT-PCR analysis and expressed in relation to ß-actin. Error bars in (A) and (B) indicate SD of triplicate determinations. [View Larger Version of this Image (13K GIF file)]
 

Figure 4 Fig. 4.. Role of adenosine and P1 receptors in neutrophil migration. (A) The effect of exogenous adenosine added to the lower or upper wells on neutrophil chemotaxis was assessed with the trans-well assay in the presence (circles) or absence (diamonds) of FMLP. (B) Effect of ADA on FMLP-induced chemotaxis. (C) Effects of the A3-receptor–selective agonist N(6)-(3-iodobenzyl) adenosine-5'-N-methylcarboxamide (IB-MECA) and of the A2- and A1-receptor–selective agonists 2-p-[2-carboxyethyl] phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS 21680) and N6-cyclopentyladenosine (CPA), respectively, on chemotaxis toward FMLP. (D) Effects of A3-receptor–selective antagonist MRS 1191 and antagonists of other P1 receptors on chemotaxis toward FMLP. (E) Composite images and cell migration traces of cells migrating toward a micropipette tip containing 100 nM FMLP in the absence or presence of 10 U/ml of ADA or 10 µM MRS 1191. Error bars in (A) to (D) indicate SD of triplicate determinations. [View Larger Version of this Image (53K GIF file)]
 

Figure 5 Fig. 5.. Localization of A3 receptors to the leading edge of migrating cells. (A) The cell surface expression of A3 receptors of human neutrophils at different time points after stimulation of cells with 100 nM FMLP was assessed with flow cytometry and a primary antibody recognizing an extracellular domain of the receptor. Error bars indicate SD of triplicate determinations. (B) Fluorescent image of an unstimulated HL60 cell expressing an A3-EGFP fusion protein. (C) HL60 cell expressing an A3-EGFP fusion protein migrating toward a micropipette tip containing 100 nM FMLP (bright field image on top). The confocal image at the bottom shows an HL60 cell migrating from the top left to the bottom right corner. (D) Colocalization of A3-EGFP fusion protein and actin in cells globally stimulated with 100 nM FMLP. [View Larger Version of this Image (44K GIF file)]
 

Figure 6 Fig. 6.. P2Y2 and A3 receptors control neutrophil chemotaxis in vitro and in vivo. (A) Migration paths of neutrophils [isolated from the bone marrow of wild-type (WT) mice and mice deficient of A3 and P2Y2 receptors] migrating toward the FMLP-receptor ligand W-peptide (100 nM). (B) Trans-well chemotaxis assays of neutrophils from A3–/–, P2Y2–/–, and WT mice toward W-peptide (100 nM). *, P < 0.0001 [analysis of variance (ANOVA) and Newman-Keuls multiple-comparison test, n = 6 mice per group]. (C) In vivo cell migration was assessed in WT mice and in A3 and P2Y2 KO mice by counting neutrophils in the peritoneal cavity 4 hours after intraperitoneal injection of 108 living Staphylococcus bacteria (solid bars) or vehicle (open bars). *, P < 0.0001 (ANOVA and Newman-Keuls multiple-comparison test, n = 6 animals per group). (D) Roles of ATP release, P2Y2, and A3 receptors in neutrophil chemotaxis: Activation of the formyl-peptide receptor (FPR) stimulates localized ATP release, resulting in activation of nearby P2Y2 receptors that amplify chemotactic signals and gradient sensing by stimulating the production of phosphoinositide 3-kinase (PI3K), phosphatidylinositol 3,4,5-trisphosphate (PIP3), and recruitment of Rac, Cdc42, and F-actin to the leading edge. Translocation of A3 receptors to the leading edge, adenosine (ADO) formation by ecto-ATPases/nucleotidases (EN), and autocrine activation of A3 receptors facilitate directed migration. Error bars in (B) and (C) indicate SD. [View Larger Version of this Image (24K GIF file)]
 


To Advertise     Find Products


Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882