Multi-Inhibitory Effects of A2A Adenosine Receptor Signaling on Neutrophil Adhesion Under Flow.

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J Immunol. Author manuscript; available in PMC 2016 October 15. Published in final edited form as: J Immunol. 2015 October 15; 195(8): 3880–3889. doi:10.4049/jimmunol.1500775.

Multi-inhibitory effects of A2A adenosine receptor signaling on neutrophil adhesion under flow** Tadayuki Yago*, Hiroki Tsukamoto†,‡, Zhenghui Liu*, Ying Wang*,§, Linda F. Thompson†,¶,#, and Rodger P. McEver*,§,# *Cardiovascular

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†Immunobiology

Biology Program, Oklahoma Medical Research Foundation, Oklahoma City, OK and Cancer Program, Oklahoma Medical Research Foundation, Oklahoma City,

OK ‡Laboratory

of Oncology, Pharmacy Practice and Sciences, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan §Department

of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK ¶Department

of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK

Abstract Author Manuscript

A2A adenosine receptor (A2AAR) signaling negatively regulates inflammatory responses in many disease models, but the detailed mechanisms remain unclear. We used the selective A2AAR agonist, ATL313, to examine how A2AAR signaling affects human and murine neutrophil adhesion under flow. Treating neutrophils with ATL313 inhibited selectin-induced, β2 integrindependent slow rolling and chemokine-induced, β2 integrin-dependent arrest on ICAM-1. ATL313 inhibited selectin-induced β2 integrin extension, which supports slow rolling, and chemokine-induced hybrid domain “swing-out”, which supports arrest. Furthermore, ATL313 inhibited integrin outside-in signaling as revealed by reduced neutrophil superoxide production and spreading on immobilized anti-β2 integrin antibody. ATL313 suppressed selectin-triggered activation of Src family kinases (SFKs) and p38 MAPK, chemokine-triggered activation of Rasrelated protein 1 (Rap1), and β2 integrin-triggered activation of SFKs and Vav cytoskeletal regulatory proteins. ATL313 activated protein kinase A (PKA) and its substrate C-terminal Src kinase (Csk), an inhibitor of SFKs. Treating neutrophils with a PKA inhibitor blocked the actions of ATL313. In vivo, ATL313-treated neutrophils rolled faster and arrested much less frequently in postcapillary venules of the murine cremaster muscle after TNF-α challenge. Furthermore,

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**This work was supported by National Institutes of Health grants HL034363 to R.P.M. and AI18220 to L.F.T. #

Co-corresponding author: Address correspondence to: Linda F. Thompson, Ph.D., Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, 825 N.E. 13th Street, Oklahoma City, OK 73104; [email protected]; or Rodger P. McEver, M.D., Cardiovascular Biology Program, Oklahoma Medical Research Foundation, 825 N.E. 13th Street, Oklahoma City, OK 73104; [email protected]. Authorship T.Y., H.T., Z.L., and Y.W. performed research; T.Y., H.T., Z.L., Y.W., R.P.M. and L.F.T. analyzed data; T.Y., R.P.M. and L.F.T. designed research and wrote the paper. R.P.M. has equity interest in Selexys Pharmaceuticals Corporation. The other authors declare no conflicting financial interests.

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ATL313 markedly suppressed neutrophil migration into the peritoneum challenged with thioglycollate. ATL313 did not affect A2AAR-deficient neutrophils, confirming its specificity. Our findings provide new insights into the anti-inflammatory mechanisms of A2AAR signaling and the potential utility of A2AAR agonists in inflammatory diseases.

Introduction

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Adenosine is a short-lived endogenous nucleoside that governs cellular functions by signaling through G-protein-coupled adenosine receptors (ARs)††. Four ARs have been identified: A1, A2A, A2B, and A3, which differ in tissue distribution, affinity for adenosine, and downstream signaling pathways (1). Neutrophils express all four ARs (2). The A2AAR has high affinity for adenosine and is also expressed on mast cells, monocytes, macrophages, eosinophils, T cells, and platelets (3). Suppressive effects of the A2AAR on leukocyte function in vitro and in vivo have been described (4). A2AAR agonists inhibit human neutrophil activation (5–7) and reduce cytokine production induced by T-cell receptor engagement (8). A2AAR agonists inhibit neutrophil adhesion and infiltration, inflammatory cytokine production, neutrophil degranulation, and oxidative burst (4). Mice lacking A2AARs exhibit increased leukocyte emigration and tissue damage in diverse models of inflammation (9–12), and augmented neutrophil recruitment and increased neointimal formation in atherosclerotic arteries after injury (13). A2AAR agonists have antiinflammatory actions in many disease models, including chronic obstructive pulmonary disease, ischemic heart disease, arthritis, sepsis, and inflammatory bowel disease (1). A2AAR signaling is thought to contribute to the anti-inflammatory effects of the adenosineelevating drug, methotrexate, in patients with rheumatoid arthritis (1). A2AAR activation reduces neutrophil-dependent inflammation and oxidative activity by protein kinase A (PKA)-dependent mechanisms (14, 15). However, the detailed molecular mechanisms by which A2AARs control adhesion and migration of immune cells, especially neutrophils, remain unclear.

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Neutrophil migration into inflamed sites is controlled by sequential adhesive and signaling events (16). First, neutrophils tether to and roll on P- and E-selectin on activated endothelial cells. Engagement of P-selectin glycoprotein ligand-1 (PSGL-1) by E- or P-selectin or CD44 by E-selectin triggers a signaling pathway that activates, among other mediators, Src family kinases (SFKs), ITAM-containing adaptor proteins, spleen tyrosine kinase (Syk), Bruton’s tyrosine kinase, Src homology domain-containing protein of 76 kDa (SLP-76), p38 MAPK, and the GTPase Ras-related protein 1a (Rap1a) (17–21). Rap1a, through effectors, recruits talin1 to the β2 integrin cytoplasmic tail, converting the ectodomain of integrin αLβ2 from a bent to an extended, “intermediate-affinity” conformation that slows rolling velocities through reversible interactions with ICAM-1 (22, 23). Rolling neutrophils encounter immobilized chemokines such as CXCL1, which engage Gαi-coupled receptors to trigger a signaling pathway that activates, among other mediators, phosphatidylinositol 3-kinase (PI3K), phospholipase C (PLC), and the small GTPases Rac, RhoA, and Rap1a (24, 25). ††Nonstandard abbreviations AR, adenosine receptor; Csk, C-terminal Src kinase; PKA, protein kinase A; PSGL-1, P-selectin glycoprotein ligand-1; Rap1, Rasrelated protein 1; SFK, Src family kinase, Syk, spleen tyrosine kinase J Immunol. Author manuscript; available in PMC 2016 October 15.

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Chemokine signaling recruits both talin1 and kindlin-3 to β2 integrin tails, which converts the ectodomain of integrin αLβ2 to an extended, “high-affinity” conformation that arrests rolling cells on ICAM-1 (22, 23). In turn, binding of ICAM-1 to high-affinity αLβ2 rapidly triggers “outside-in” signaling that activates SFKs, ITAM motif-containing adaptors, Syk, SLP-76, and Vav guanine nucleotide-exchange proteins that rearrange the actin cytoskeleton (26, 27). Outside-in signaling enhances cell spreading, adhesion strengthening, and migration (28). Whether A2AAR engagement perturbs one or more of these pathways to inhibit neutrophil adhesion is unknown. In this study, we report that the A2AAR agonist, ATL313, inhibits neutrophil adhesion and migration by suppressing, through PKA, both integrin inside-out and outside-in signaling under flow. These data explain how A2AAR signaling limits neutrophil infiltration into inflammatory sites.

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Materials and methods Reagents

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Human P-selectin purified from platelets (29) and recombinant soluble human E-selectin (30) and murine P-selectin-IgM, E-selectin-IgM, and control CD45-IgM chimeric molecules (31) were previously described. Recombinant human ICAM-1 and IL-8 and murine ICAM-1 IgG Fc, CXCL1, tumor necrosis factor (TNF)-α, and murine anti-human ICAM-1 mAb (clone BBIG-I1) were from R&D Systems. Murine mAbs against human CD18 (β2 integrin subunit, clone MEM148), human Rap1 (crossreacts with murine Rap1), and human Rap1GTP (crossreacts with murine Rap1-GTP) and rabbit polyclonal antibodies against Rap1 and phospho-Csk (S364) were from Abcam. Hybridomas producing murine mAbs against human β2 integrin subunit (clones IB4 and KIM127) were from the American Type Culture Collection. Rat anti-murine β2 integrin mAb GAME-46, rat anti-murine ICAM-1 mAb, and Alexa 488-labeled rat anti-murine CD31 mAb were from BD Biosciences. Rabbit antibodies to SFK, phospho-SFK (Y416), phospho-Syk (Y519/520), p38 MAPK, phospho-p38 MAPK, AKT, and phospho-PKA (T197), goat horseradish peroxidase-conjugated anti-rabbit IgG, and cAMP-dependent PKA inhibitor H89 were from Cell Signaling Technology. Rabbit anti-phospho-Vav (Y174) antibody and murine anti-β-actin mAb (reacts with murine βactin) were from Santa Cruz Biotechnology. Goat horseradish peroxidase-conjugated antimouse IgG and Protein A/G agarose beads were from Thermo Scientific. PKH26 and PKH67 dyes were from Sigma-Aldrich. Isotype control murine IgG1 and rat IgG1 and phycoerythrin (PE)-conjugated goat anti-mouse IgG were from Invitrogen. ATL313, a selective A2AAR agonist (32), was supplied by Adenosine Therapeutics LLC. It was dissolved in DMSO at 10 mM and then diluted in Hanks’ balanced salt solution (HBSS) at 100 nM for in vitro studies. An equivalent concentration of DMSO in HBSS (1:105) was used as a negative control.

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Mice Wild-type (WT) C57BL/6 mice were from The Jackson Laboratory. A2AAR−/− mice (33) on the C57BL/6 background were generous gifts from Jiang-Fan Chen (Boston University).

J Immunol. Author manuscript; available in PMC 2016 October 15.

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Protocols were approved by the Institutional Animal Care and Use Committee of the Oklahoma Medical Research Foundation. Cells Murine bone marrow leukocytes were isolated by flushing femurs and tibias with 10 ml HBSS without Ca2+ or Mg2+ as described previously (19, 34). After red blood cell lysis, the leukocytes were resuspended at 106/ml in HBSS containing 1.26 mM Ca2+, 0.81 mM Mg2+, and 0.5% human serum albumin (HSA). Murine neutrophils were isolated from bone marrow leukocytes by a density gradient method (35). Human neutrophils were isolated as described previously (29). Flow cytometry

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Flow cytometry was performed as described previously (36). Human neutrophils were pretreated with 100 nM ATL313 or DMSO (1:105 in HBSS) for 30 min at room temperature and then incubated with 50 μg/ml human platelet-derived P-selectin with or without 5 mM EDTA for 30 min. In some experiments, neutrophils were pre-incubated with 100 nM ATL313 or DMSO (1:105 in HBSS) for 30 min at room temperature and then stimulated with 2 nM IL-8 for 10 min. The cells were stained with 10 μg/ml isotype control mouse IgG, IB4, KIM127, or MEM148, followed by PE-conjugated goat anti-mouse IgG. Data were acquired on a FACSCalibur instrument (BD Biosciences) and analyzed with Cell Quest software. Flow chamber assay


Adhesion of human neutrophils or murine bone marrow leukocytes under flow was studied as previously described (19, 36, 37). For human neutrophils, 10 μg/ml ICAM-1, KIM127, MEM148, or isotype control murine IgG was co-immobilized with 1 μg/ml human Pselectin or 10 μg/ml human E-selectin with or without 10 μg/ml IL-8. For murine bone marrow leukocytes, 20 μg/ml ICAM-1 IgG Fc was absorbed with or without 10 μg/ml CXCL1. Murine P-selectin-IgM or E-selectin-IgM was captured by immobilized anti-human IgM Fc mAb. The plates were then blocked with 1% HSA. Human neutrophils or murine bone marrow leukocytes were pre-incubated with 100 nM ATL313 or with DMSO (1:105) for 30 min at room temperature. The treated cells (106/ml in HBSS with Ca2+, Mg2+, and 0.5% HSA) were perfused over the substrates at a wall shear stress of 1 dyn/cm2. In some dishes, the function of immobilized ICAM-1 was blocked by adding anti-human or antimurine ICAM-1 mAb (20 μg/ml). In some experiments, murine bone marrow leukocytes were pre-treated with 10 μM H89 20 min before ATL313 treatment. Rolling and arrested cells were analyzed using a videomicroscopy system coupled to a digital analysis system with Element software (Nikon). Arrested cells were scored as “round” (round and bright) or “spread” (irregular and dark). F(ab′)2 fragments of IB4 or GAME-46 were generated with an F(ab′)2 preparation kit (Thermo Scientific), following the manufacturer’s protocol. Human neutrophils, or murine bone marrow leukocytes from wild type (WT) or A2AAR−/− mice, each at 106 cells/ml, were introduced into 35-mm culture dishes pre-coated with 20 μg/ml F(ab′)2 fragments of IB4 (for human neutrophils) or GAME-46 (for murine cells) at 0.25 dyn/cm2 and allowed to


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accumulate by stopping the flow. After 15 min, unbound cells were washed out with buffer at 0.25 dyn/cm2, and then non-spread (round and bright) and spread (irregular and dark) cells were counted using Element software. In some experiments, human neutrophils or murine bone marrow leukocytes were preincubated with 100 nM ATL313 or with DMSO (1:105 in HBSS) for 30 min at room temperature. In other experiments, murine bone marrow leukocytes were pre-treated with 10 μM H89 20 min before ATL313 treatment. Signaling assays


To study selectin-induced signaling, bone marrow leukocytes from WT or A2AAR−/− mice were incubated in 6-well plates with P-selectin IgM Fc captured on immobilized anti-human IgM Fc antibody on a rotary shaker at 65 rpm for 10 min at room temperature as previously described (19). To analyze signaling induced by engagement of β2 integrins, bone marrow leukocytes or isolated murine neutrophils were incubated in 6-well plates coated with 20 μg/ml (Fig. 4) or 50 μg/ml (Fig. 5) of F(ab′)2 fragments of GAME-46 or isotype control rat IgG for 10 min at room temperature as previously described (38). For chemokine signaling, cells were stimulated with 100 nM CXCL1 for 10 min. After the above treatments, the cells were lysed with 1% Triton X-100, 125 mM NaCl, 50 mM Tris pH 8.0, 10 mM EDTA, 2 mM PMSF, 0.1% SDS with a protease and phosphatase inhibitor cocktail (Thermo Scientific). The cell lysates were analyzed by Western blotting using rabbit antibodies against SFK, phospho-SFK (Y416), p38, phospho-p38, AKT, phospho-AKT, phospho-Vav, phospho-PKA, phospho-Csk, and β-actin. To examine activation of Rap1 induced by CXCL1, 200-μl lysates were incubated with protein A/G agarose for 3 h at 4°C and centrifuged. The pre-cleared lysates were incubated with murine anti-Rap1 or anti-Rap1GTP antibody at 4°C overnight and then with protein A/G agarose for 3 h at 4°C. After washing the beads, bound Rap1 was eluted with 200 μl boiling SDS-PAGE sample buffer. Twenty μl of immunoprecipitated lysate was analyzed by Western blotting using rabbit polyclonal anti-Rap1 antibody. Superoxide production was measured as described previously (39). Briefly, isolated murine neutrophils were pretreated with DMSO, ATL313, or ATL313 with H89. The cells (2 × 106 in 100 μl) were then incubated in 6-well plates coated with 50 μg/ml of F(ab′)2 fragments of GAME-46 or isotype control IgG with 10 nmol ferricytochrome C (Fe3+, Sigma-Aldrich) for 1 h at 37°C. Absorbance at 550 nm was measured in a FLUOstar Omega microplate reader (BMG Labtech) to calculate reduced cytochrome C (Fe2+). In some experiments, 50 U/well of superoxide dismutase (Sigma-Aldrich) was added to confirm that reduction of cytochrome C was caused by O2- production. In other experiments, total cytochrome C in each well was reduced by adding 10 μg/well of sodium dithionite (Sigma-Aldrich).

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Spinning-disk intravital microscopy Spinning-disk intravital microscopy was performed as previously described (40). A2AAR−/− mice were anesthetized by intraperitoneal injection of 1.25% Avertin. The cremaster muscle was exteriorized and superfused with thermocontrolled (35°C) bicarbonate-buffered saline 4 h after intrascrotal injection of 500 ng murine TNF-α (31). Bone marrow leukocytes from WT or A2AAR−/− mice were labeled with red fluorescent dye (PHK 26, Sigma-Aldrich) or far-red fluorescent dye (CellVue Claret, Sigma-Aldrich), and then pre-incubated with 100


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nM ATL313 or DMSO (1:105 in HBSS) for 30 min at room temperature. Immediately after exteriorization of the cremaster muscle, the differentially labeled cells were resuspended in saline in a 1:1 mixture (108/ml), and 0.2 ml was injected intravenously. The vessels were labeled by intravenous injection of Alexa 488-labeled anti-CD31 mAb. In some experiments, 20 μg anti-P-selectin mAb and anti-β2 integrin mAb were sequentially injected intravenously. The microcirculation of the cremaster muscle was visualized using a Nikon ECLIPSE E600-FN upright microscope equipped with an Olympus 20 x/0.95W XLUM Plan Fl water immersion objective lens and a motorized piezo z-stage (MFC-2000, Applied Scientific Instrumentation). The microscope was coupled to a confocal light path (Solamere Technology Group) based on a modified Yokagawa CSU-X1 head (Yokagawa Electric Corporation). Three lasers with excitation at 488, 561, and 642 nm (Coherent) were rapidly and sequentially selected by an acousto-optic tunable filter, merged into a single optic cable and introduced into the CSU-X1 head. Fluorescence signals were detected through the appropriate emission filters of ET525/50, ET605/52, and ET700/75 controlled by an ASI FW-1000 Filterwheel (Applied Scientific Instrumentation). A 512 × 512 pixel back-thinned EMCCD camera (C9100-13, Hamamatsu) was used for acquisition of the fluorescent images. The NIH acquisition software Micromanager was used to drive the spinning-disk confocal microscope and captured images. The images were analyzed with ImageJ software.

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Competitive neutrophil recruitment assay

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Competitive neutrophil recruitment was measured as described (19). Bone marrow leukocytes from WT or A2AAR−/− mice treated with DMSO or ATL313 were labeled with red (PKH26) or green (PKH67) dye. Red and green cells were resuspended in HBSS at 108 cells/ml and mixed at a 1:1 ratio. Recipient A2AAR−/− mice were injected with 1 ml of 4% thioglycollate intraperitoneally and with 200 μl of the labeled cell mixture retroorbitally. After 4 h, blood was collected. The mice were sacrificed, and peritoneal cells were collected with 10 ml PBS containing 0.1% BSA and 5 mM EDTA. Neutrophils in blood and peritoneal lavage (identified by light scattering and Ly6G expression using flow cytometry) were counted. The data were plotted as the ratio of PKH26-labeled neutrophils from the indicated population compared to PKH67-labeled A2AAR−/− neutrophils in blood or peritoneal lavage. Equivalent results were obtained after reversing the dyes to label the respective cell populations. Statistics Data are expressed as mean ± SEM. Comparisons used the Student’s t-test (unpaired and two tailed). P < 0.05 was considered to be significant.

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Results Treating human neutrophils with the A2AAR agonist, ATL313, inhibits selectin-induced, β2 integrin-mediated slow rolling on ICAM-1, chemokine-induced, β2 integrin-mediated arrest and spreading on ICAM-1, and spreading on anti-β2 integrin antibody We treated human neutrophils with control DMSO vehicle or the A2AAR-specific agonist ATL313 at an optimal concentration established in previous studies (8). Control or ATL313treated neutrophils were perfused over immobilized human P- or E-selectin with or without


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co-immobilized human ICAM-1 (Fig. 1A, B). Control neutrophils rolled slower on P- or Eselectin and ICAM-1 than on P- or E-selectin alone. Anti-ICAM-1 mAb prevented slower rolling, consistent with its dependence on integrin αLβ2 interactions with ICAM-1 (19). In contrast, ATL313-treated neutrophils rolling on P- or E-selectin failed to roll slower on coimmobilized ICAM-1 (Fig. 1A, B). Thus, the A2AAR agonist blocks selectin-induced, β2 integrin-dependent slow rolling on ICAM-1.

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We next perfused control or ATL313-treated neutrophils over P- or E-selectin coimmobilized with both ICAM-1 and the human chemokine IL-8. Most rolling control neutrophils rapidly arrested (firm adhesion), and many of the arrested cells transitioned from a round to a spread appearance (Fig. 1C, D). Arrest on ICAM-1 reflects IL-8-mediated inside-out signaling that converts integrin αLβ2 to an extended, high-affinity conformation (22). The bound ICAM-1 then induces spreading through integrin outside-in signaling (28). Significantly less ATL313-treated neutrophils converted from rolling to arrest, and only a few arrested cells spread (Fig. 1C, D). These data demonstrate that the A2AAR agonist blocks chemokine-induced, β2 integrin-dependent arrest on ICAM-1 and suggest that it also blocks β2 integrin outside-in signaling. To further test ATL313 effects on outside-in signaling, we incubated neutrophils with immobilized F(ab′)2 fragments of anti-β2 integrin mAb (Fig. 1E). Almost all control neutrophils spread, due to integrin outside-in signaling triggered by bound mAb, whereas very few ATL313-treated neutrophils spread. Thus, the A2AAR agonist inhibits chemokine-induced, β2 integrin-mediated arrest on ICAM-1 and β2 integrin-induced spreading on ICAM-1 or anti-β2 integrin mAb.

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Treating human neutrophils with the A2AAR agonist, ATL313, prevents selectin- and chemokine-induced β2 integrin extension and chemokine-induced hybrid-domain “swingout” The mAb KIM127 binds to an epitope near the genu (knee) of the human β2 subunit, which is exposed after integrin extension (41). The mAb MEM148 reports “swing-out” of the hybrid domain from the βI domain after extension of the human β2 subunit; hybrid-domain swing-out is associated with high affinity for ligand (42). Soluble, oligomeric human Pselectin induced epitopes for KIM127 but not MEM148 on human neutrophils, and did not alter binding of mAb IB4 to an activation-insensitive epitope on human β2 (Fig. 2A). Blocking Ca2+-dependent binding of soluble P-selectin to PSGL-1 with EDTA prevented KIM127 binding. Treating neutrophils with ATL313 suppressed P-selectin-induced binding of KIM127 (Fig. 2A). Soluble chemokine IL-8 induced epitopes for both KIM127 and MEM148 but did not alter binding of IB4 (Fig. 2B). Treating neutrophils with ATL313 suppressed binding of both KIM127 and MEM148 (Fig. 2B).

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Human neutrophils rolling on P-selectin arrested on co-immobilized KIM127, and they arrested on MEM148 when IL-8 was also co-immobilized (Fig. 2C). Neutrophils rolling on P-selectin did not arrest on isotype control IgG. ATL313 treatment inhibited selectin- and chemokine-induced neutrophil arrest on KIM127 and chemokine-induced arrest on MEM148, supporting the flow cytometry data. Taken together, these data indicate that the A2AAR agonist prevents selectin- and chemokine-induced integrin extension and chemokine-induced hybrid-domain swing-out.


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A2AAR signals through PKA in murine leukocytes to inhibit selectin-induced, β2 integrinmediated slow rolling on ICAM-1, chemokine-induced, β2 integrin-mediated arrest on ICAM-1, and spreading on anti-β2 integrin antibody

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We next treated bone marrow leukocytes from WT or A2AAR−/− mice with control DMSO or ATL313 and perfused them over immobilized murine P- or E-selectin with or without coimmobilized murine ICAM-1 and the murine chemokine CXCL1. Greater than 90% of leukocytes rolling on selectins were neutrophils, as documented by nuclear morphology of stained adherent cells and by reactivity with anti-Ly6G mAb (data not shown), consistent with previous studies (19, 37). Flow cytometry revealed that >90% of bone marrow leukocytes expressing CXCR2, the receptor for CXCL1, also expressed Ly6G (data not shown). Thus, virtually all bone marrow leukocytes interacting with P-selectin and CXCL1 in flow-chamber assays were Ly6G-positive neutrophils. Like human neutrophils, murine neutrophils from both genotypes rolled slower on murine P- or E-selectin and ICAM-1 than on P- or E-selectin alone (Fig. 3A, B). Anti-ICAM-1 mAb prevented integrin-dependent slow rolling. ATL313 blocked slow rolling of neutrophils from WT mice but not from A2AAR−/− mice, confirming the specificity of the agonist for the A2AAR. Neutrophils from both genotypes rolling on P- or E-selectin rapidly arrested and spread on ICAM-1 when the chemokine CXCL1 was co-immobilized (Fig. 3C, D). ATL313 inhibited arrest and spreading of neutrophils from WT but not A2AAR−/− mice. Similarly, ATL313 inhibited spreading of leukocytes from WT but not A2AAR−/− mice on anti-β2 integrin F(ab′)2 (Fig. 3E). A2AAR signaling increases cAMP, which activates PKA (43, 44). H89, an inhibitor of PKA, blocked the effects of ATL313 on slow rolling, arrest, and spreading (Fig. 3F–H). These data demonstrate that A2AAR acts through PKA to inhibit β2 integrin-dependent slow rolling, arrest, and spreading of leukocytes, including neutrophils.

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A2AAR signaling in murine leukocytes inhibits selectin- or β2 integrin-induced activation of SFKs, p38 MAPK, or Vav, and chemokine-induced activation of Rap1

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To investigate signaling pathways affected by A2AAR activation, we incubated WT or A2AAR−/− leukocytes on immobilized P-selectin or anti-β2 integrin F(ab′)2, or with fluidphase CXCL1. Immobilized P-selectin-IgM Fc, but not control CD45-IgM Fc, induced phosphorylation of SFKs and a downstream target, p38 MAPK (Fig. 4A). Blocking Ca2+dependent binding of P-selectin with EDTA prevented phosphorylation of SFKs and p38 MAPK. ATL313 inhibited phosphorylation of SFKs and p38 MAPK in WT but not A2AAR−/− leukocytes. Immobilized anti-β2 integrin F(ab′)2, but not isotype-control F(ab′)2, induced phosphorylation of SFKs and a downstream target, Vav (Fig. 4B). ATL313 inhibited phosphorylation of SFKs and Vav in WT but not A2AAR−/− leukocytes. CXCL1 induced phosphorylation of AKT, a key substrate of PI3K (Fig. 4C). ATL313 did not inhibit AKT phosphorylation in WT or A2AAR−/− leukocytes, indicating that A2AAR signaling does not affect chemokine-induced activation of PI3K/AKT. However, ATL313 did inhibit CXCL1-induced activation of Rap1 to its GTP-bound form, a downstream consequence of PLC signaling (45), in WT but not A2AAR−/− leukocytes (Fig. 4D). The PKA inhibitor H89 blocked the effects of ATL313 (Fig. 4E–G). These data demonstrate that A2AAR acts through PKA to inhibit multiple signaling pathways in leukocytes, including neutrophils.


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A2AAR signaling in murine neutrophils activates PKA and Csk and inhibits β2 integrininduced Syk activation and superoxide production To study β2 integrin signaling specifically in murine neutrophils, we isolated neutrophils from bone marrow leukocytes. Neutrophils plated on anti-β2 integrin F(ab′)2, but not isotype control F(ab′)2, activated SFKs and a downstream target, Syk, (Fig. 5A). ATL313 blocked activation of SFKs and Syk, which was restored in neutrophils treated with the PKA inhibitor H89. Consistent with these findings, ATL313 directly activated PKA and its substrate C-terminal Src kinase (Csk), an inhibitor of SFKs (46). H89 blocked these effects of ATL313 (Fig. 5A). These results further demonstrate that the A2AAR acts directly through PKA, and likely its effector Csk, to suppress signaling of SFKs and downstream mediators such as Syk.

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Neutrophils plated on anti-β2 integrin F(ab′)2, but not control F(ab′)2, also generated superoxide (Fig. 5B). ATL313 blocked superoxide production, which was restored in neutrophils treated with H89. Thus, ATL313 acts through PKA to inhibit distinct β2 integrin-dependent effector responses in neutrophils. A2AAR signaling in murine leukocytes inhibits β2 integrin-mediated slow rolling and arrest in vivo


To determine whether A2AAR regulates leukocyte adhesion in vivo, we used spinning-disk microscopy to measure leukocyte rolling and arrest in venules of the cremaster muscle 4 h after stimulation with TNF-α, which increases expression of P-selectin, E-selectin, and CXCL1 (47). In this model, almost all rolling and arrested leukocytes are neutrophils (19, 47, 48). WT or A2AAR−/− leukocytes were treated with DMSO or ATL313, labeled with different fluorescent dyes, mixed in a 1:1 ratio, and injected intravenously into A2AAR−/− mice to restrict A2AAR signaling to the transfused cells. Ten minutes after injection, rolling velocities were measured in the same venules, labeled with fluorescent anti-CD31 mAb, before and after sequentially injecting blocking mAbs to P-selectin and β2 integrins. Anti-Pselectin mAb did not alter velocities, consistent with the dominance of E-selectin for controlling rolling in this model (17, 47, 49). DMSO-treated WT and A2AAR−/− leukocytes rolled with comparable velocities (Fig. 6A and Supplemental Video 1). Anti-β2 integrin mAb similarly increased velocities, revealing the integrin-dependent component of slow rolling (Fig. 6A). However, ATL313-treated WT leukocytes rolled faster than ATL313treated A2AAR−/− leukocytes (Fig. 6A and Supplemental Video 2). Anti-β2 integrin mAb increased the velocities of ATL313-treated A2AAR−/− leukocytes but did not further increase the velocities of ATL313-treated WT leukocytes (Fig. 6A). In separate experiments, we measured the number of arrested (firmly adherent) labeled leukocytes 90 minutes after injection. ATL313 treatment significantly reduced arrest of WT but not A2AAR−/− leukocytes (Fig. 6B). Finally, we measured competitive migration of differentially labeled leukocytes into the thioglycollate-challenged peritoneum, which is mediated by selectins, chemokines, and β2 integrins (48, 50, 51). ATL313 treatment markedly reduced migration of WT but not A2AAR−/− neutrophils, identified by light scattering and Ly6G expression using flow cytometry (Fig. 6C). These data demonstrate that the A2AAR agonist inhibits selectin-induced, β2 integrin-dependent slow rolling and chemokine-induced, β2 integrin-dependent arrest and migration in vivo.


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Discussion Our data define mechanisms by which the A2AAR negatively regulates neutrophil adhesion (Fig. 7). The A2AAR agonist ATL313 inhibited key events in selectin- and chemokineinduced signaling that activate β2 integrins, and in β2 integrin outside-in signaling. ATL313 inhibited selectin-induced β2 integrin extension, which supports slow rolling, and chemokine-induced hybrid domain “swing-out”, which supports arrest (Fig. 2). ATL313treated neutrophils rolled faster and arrested less efficiently on ICAM-1 in vitro and in vivo, and spread poorly after they arrested (Figs. 1, 3, and 6). ATL313 inhibited integrindependent adhesion through PKA, because the PKA inhibitor H89 blocked the agonist’s effects (Figs. 3 and 4).


In neutrophils, ATL313 blocked the earliest known step in selectin signaling and integrin outside-in signaling: the activation of SFKs (Figs. 4 and 5). The signaling cascade triggered by selectin and integrin engagement resembles that of canonical immunoreceptors such as the T-cell receptor (28, 52, 53). ATL313 activated PKA and its substrate, Csk (Fig. 5A). PKA phosphorylation of Csk suppresses T-cell receptor-induced activation of the SFK Lck in T cells (46), and Csk-deficient neutrophils exhibit increased adhesion after crosslinking integrin αMβ2 (54). Thus, ATL313 may trigger PKA phosphorylation of Csk to inhibit selectin- or integrin-induced activation of SFKs in neutrophils. Blockade of SFKs inhibits all downstream signaling steps, including phosphorylation of Syk in neutrophils and the related ZAP70 in T cells (28, 53). Indeed, ATL313, through PKA, blocks T-cell receptor-induced phosphorylation of ZAP70 (8). We found that ATL313 blocked selectin-induced downstream phosphorylation of SFKs and p38 MAPK (Fig. 4), which leads to integrindependent slow rolling (19, 21). It also blocked integrin-induced downstream activation of Vav proteins (Fig. 4), which rearrange the actin cytoskeleton to initiate spreading and strengthen adhesion (26). Our data are consistent with the ability of elevated cAMP to inhibit integrin-dependent spreading of TNF-α-stimulated neutrophils (55).

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ATL313 blocked chemokine-induced conformational change in β2 integrins that is associated with high affinity for ICAM-1 (Fig. 2). As a result, ATL313 significantly impaired neutrophil arrest on ICAM-1 under flow (Figs. 1, 3, and 6) and β2 integrindependent migration into the inflamed peritoneum (Fig. 6). These effects are consistent with reports that cAMP or PKA inhibits chemokine-mediated integrin activation in neutrophils (55, 56). ATL313 did not prevent chemokine activation of PI3K (Fig. 4). PI3K signals do not alter αLβ2 affinity for ICAM-1, but do enhance avidity by increasing the integrin’s lateral mobility (57). Instead, ATL313 blocked chemokine activation of the small GTPase Rap1 to its GTP-bound form (Fig. 4). GTP-Rap1, through effectors such as Rap1-GTPinteracting molecule, activates integrins by recruiting talin to integrin β tails (58). It also recruits RAP ligand to αL tails (24). ATL313-generated PKA may inhibit Rap1 activation in neutrophils by complementary mechanisms. In transfected cells, PKA phosphorylation of GTP-Rap1 removes it from the membrane and accelerates its inactivation by hydrolysis of GTP (59). In platelets, PKA phosphorylation of the guanine nucleotide-exchange protein CalDAG-GEF1 inhibits its ability to load GTP onto Rap1b (60). ATL313 likely also directly inhibits activation of Rap1 in neutrophils after selectin or integrin signaling. In neutrophils, ATL313-generated cAMP, which activates PKA (43, 44), negatively regulated Rap1-


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dependent activation of β2 integrins (Fig. 3). In some settings, by contrast, cAMP directly activates a Rap1 guanine nucleotide-exchange protein in a PKA-independent manner (61, 62). We found that the A2AAR agonist ATL313 inhibits critical steps in both integrin inside-out and outside-in signaling, with important consequences for integrin-dependent neutrophil adhesion in vitro and in vivo, and for effector responses such as superoxide production (Fig. 5). These actions provide mechanistic insights into A2AAR functions in diverse models of neutrophil-mediated inflammation (1, 9–15). Further studies of A2AAR signaling may lead to more effective adenosine-based therapies for inflammatory diseases.

Supplementary Material Refer to Web version on PubMed Central for supplementary material.

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Acknowledgments We thank Stephanie McGee for technical assistance, Dr. Jiang-Fan Chen for providing A2AAR−/− mice, and Dr. Joel Linden for constructive comments on the manuscript.

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Figure 1. Treating human neutrophils with the A2AAR agonist, ATL313, inhibits selectininduced, β2 integrin-mediated slow rolling on ICAM-1, chemokine-induced, β2 integrinmediated arrest and spreading on ICAM-1, and spreading on anti-β2 integrin antibody

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(A) Velocities of human neutrophils rolling on P-selectin with or without co-immobilized ICAM-1, in the presence or absence of anti-ICAM-1 mAb. (B) Velocities of human neutrophils rolling on E-selectin with or without co-immobilized ICAM-1, in the presence or absence of anti-ICAM-1 mAb. (C) Numbers of human neutrophils rolling, arrested and round, or arrested and spread on co-immobilized P-selectin, ICAM-1, and IL-8. (D) Numbers of human neutrophils rolling, arrested and round, or arrested and spread on coimmobilized E-selectin, ICAM-1, and IL-8. (E) Numbers of adherent round or spread neutrophils on immobilized F(ab′)2 fragments of anti-human β2 integrin mAb. The shear stress in A–D was 1 dyn/cm2. The data represent the mean ± SEM from five experiments. *, P < 0.05 between indicated rolling cell populations; #, P < 0.05 between DMSO- and ATL313-treated arrested and round cells; †, P < 0.05 between DMSO- and ATL313-treated arrested and spread cells.


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Figure 2. Treating human neutrophils with the A2AAR agonist, ATL313, prevents selectin- and chemokine-induced β2 integrin extension and chemokine-induced hybrid-domain “swing-out”

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(A) DMSO- or ATL313-treated human neutrophils were incubated with or without plateletderived P-selectin in HBSS containing Ca2+ or EDTA. The cells were then incubated with isotype control mouse IgG or with anti-β2 integrin mAb IB4, KIM127, or MEM148, followed by PE-conjugated goat anti-mouse IgG. (B) DMSO- or ATL313-treated human neutrophils were incubated with IL-8. The cells were then incubated with isotype control mouse IgG or with anti-β2 integrin mAb IB4, KIM127, or MEM148, followed by PEconjugated goat anti-mouse IgG. (C) DMSO- or ATL313-treated human neutrophils were perfused over P-selectin with co-immobilized isotype control mouse IgG, KIM127, or MEM148, with or without co-immobilized IL-8. The wall shear stress was 1.0 dyn/cm2. The percentages of rolling and arrested cells are plotted. The data in A and B are representative of three experiments. The data in C represent the mean ± SEM of three experiments.


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Author Manuscript Author Manuscript Figure 3. A2AAR signals through PKA in murine leukocytes to inhibit selectin-induced, β2 integrin-mediated slow rolling on ICAM-1, chemokine-induced, β2 integrin-mediated arrest on ICAM-1, and spreading on anti-β2 integrin antibody


(A) Velocities of DMSO- or ATL313-treated leukocytes from WT or A2AAR−/− mice rolling on P-selectin, with or without co-immobilized ICAM-1, in the presence or absence of anti-ICAM-1 mAb. (B) Velocities of DMSO- or ATL313-treated leukocytes from WT or A2AAR−/− mice rolling on E-selectin, with or without co-immobilized ICAM-1, in the presence or absence of anti-ICAM-1 mAb. (C) Numbers of DMSO- or ATL313-treated leukocytes from WT or A2AAR−/− mice rolling, arrested and round, or arrested and spread on co-immobilized P-selectin, ICAM-1, and CXCL1. (D) Numbers of DMSO- or ATL313treated leukocytes from WT or A2AAR−/− mice rolling, arrested and round, or arrested and spread on co-immobilized E-selectin, ICAM-1, and CXCL1. (E) Numbers of adherent round or spread DMSO- or ATL313-treated leukocytes from WT or A2AAR−/− mice on immobilized F(ab′)2 fragments of anti-murine β2 integrin mAb. (F) Velocities of DMSO-, ATL313-, and/or H89-treated WT leukocytes rolling on P-selectin, with or without coimmobilized ICAM-1, in the presence or absence of anti-ICAM-1 mAb. (G) Numbers of DMSO-, ATL313-, and/or H89-treated WT leukocytes rolling, arrested and round, or arrested and spread on co-immobilized P-selectin, ICAM-1, and CXCL1. (H) Numbers of adherent round or spread DMSO-, ATL313-, and/or H89-treated WT leukocytes on immobilized F(ab′)2 fragments of anti-murine β2 integrin mAb. The wall shear stress in A– D, F, and G was 1 dyn/cm2. The data represent the mean ± SEM from five experiments. *, P < 0.05 between indicated rolling cell populations; #, P < 0.05 between DMSO- and


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ATL313-treated arrested and round cells; †, P < 0.05 between DMSO- and ATL313-treated arrested and spread cells.

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Author Manuscript Author Manuscript Figure 4. A2AAR signaling in murine leukocytes inhibits selectin- or β2 integrin-induced activation of SFKs, p38 MAPK, or Vav, and chemokine-induced activation of Rap1


(A) DMSO- or ATL313-treated leukocytes from WT or A2AAR−/− mice were incubated on immobilized P-selectin-IgM in the presence or absence of EDTA or on control CD45-IgM. Lysates were Western blotted with antibodies against SFKs, phospho-SFKs, p38, or phospho-p38. (B) DMSO- or ATL313-treated leukocytes from WT or A2AAR−/− mice were incubated on immobilized F(ab′)2 fragments of isotype control IgG or anti-murine β2 integrin mAb. Lysates were Western blotted with antibodies against SFKs, phospho-SFKs, phospho-Vav, or β-actin (loading control for phospho-Vav). (C) DMSO- or ATL313-treated leukocytes from WT or A2AAR−/− mice were incubated with or without CXCL1. Lysates were Western blotted with antibodies against AKT or phospho-AKT. (D) DMSO- or ATL313-treated leukocytes from WT or A2AAR−/− mice were incubated with or without CXCL1. Lysates were immunoprecipitated with anti-Rap-1 or anti-Rap1-GTP antibody on protein A/G agarose beads. Eluted proteins were Western blotted with anti-Rap1 antibody. (E) DMSO-, ATL313-, and/or H89-treated WT leukocytes were incubated on immobilized P-selectin-IgM or on control CD45-IgM. Lysates were Western blotted with antibodies against SFKs or phospho-SFKs. (F) DMSO-, ATL313-, and/or H89-treated WT leukocytes were incubated on immobilized F(ab′)2 fragments of isotype control IgG or anti-murine β2 integrin mAb. Lysates were Western blotted with antibodies against SFKs or phospho-SFKs. (G) DMSO-, ATL313-, and/or H89-treated WT leukocytes were incubated with or without CXCL1. Lysates were immunoprecipitated with anti-Rap-1 or anti-Rap1-GTP antibody on


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protein A/G agarose beads. Eluted proteins were Western blotted with anti-Rap1 antibody. The data are representative of three experiments.


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Author Manuscript Author Manuscript Figure 5. A2AAR signaling in murine neutrophils activates PKA and Csk and inhibits β2 integrin-induced Syk activation and superoxide production

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DMSO-, ATL313-, and/or H89-treated WT neutrophils were incubated on immobilized F(ab ′)2 fragments of isotype control IgG or anti-murine β2 integrin mAb. (A) Lysates were Western blotted with antibodies against phospho-PKA, phospho-Csk, phospho-SFKs, phospho-Syk, or β-actin (loading control). (B) Superoxide production was measured by a colorimetric assay. The data in A are representative of three experiments. The data in B represent the mean ± SEM from three experiments. *, P < 0.05 between indicated cell populations.


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Figure 6. A2AAR signaling in murine leukocytes inhibits β2 integrin-mediated slow rolling and arrest in vivo

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WT or A2AAR−/− leukocytes were treated with DMSO or ATL313, labeled with red (PKH26) or green (PKH67) dye, mixed in a 1:1 ratio, and injected intravenously into A2AAR−/− mice 4 h after intrascrotal injection of TNF-α. Venules were labeled with fluorescent anti-CD31 mAb. (A) Ten minutes after injection, rolling velocities of differentially labeled WT and A2AAR−/− leukocytes were measured in venules of the cremaster muscle, before and after sequentially injecting blocking mAbs to P-selectin and β2 integrins. (B) In separate experiments 90 minutes after injection, the numbers of arrested (firmly adherent), differentially labeled WT and A2AAR−/− leukocytes were measured. Representative fluorescent images of DMSO- or ATL313-treated leukocytes arrested in venules labeled with anti-CD31 mAb are shown. (C) WT or A2AAR−/− leukocytes were treated with DMSO or ATL313, labeled with PKH26 or PKH67 dye, mixed in a 1:1 ratio, and injected intravenously into A2AAR−/− mice that were also injected intraperitoneally with thioglycollate. After 4 h, neutrophils in blood and peritoneal lavage were counted. The data were plotted as the ratio of PKH26-labeled neutrophils from the indicated population


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compared to PKH67-labeled A2AAR−/− neutrophils. The data represent the mean ± SEM from four experiments (C) or five experiments (A and B). Scale bar, 10 μm.


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Figure 7. Mechanisms for A2AAR-mediated inhibition of β2 integrin inside-out and outside-in signaling in neutrophils

The A2AAR agonist ATL313 triggers synthesis of cAMP, which activates PKA. Activated PKA blocks selectin- or β2 integrin-induced activation of SFKs and chemokine-induced activation of Rap1, inhibiting β2 integrin conformational change (reported by binding of mAbs KIM127 and MEM148 to human β2 integrins) and β2 integrin-mediated slow rolling, arrest, and spreading on ICAM-1 under flow.


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Effects of adenosine A2a receptor agonist and antagonist on cerebellar nuclear factor-kB expression preceded by MDMA toxicity.

Differential effects of presynaptic versus postsynaptic adenosine A2A receptor blockade on Δ9-tetrahydrocannabinol (THC) self-administration in squirrel monkeys.

Optogenetic activation of intracellular adenosine A2A receptor signaling in the hippocampus is sufficient to trigger CREB phosphorylation and impair memory.

Transglutaminase stabilizes melanoma adhesion under laminar flow.

Overexpression of Adenosine A2A Receptors in Rats: Effects on Depression, Locomotion, and Anxiety.

2 signaling pathway.

Optogenetic activation of intracellular adenosine A2A receptor signaling in the hippocampus is sufficient to trigger CREB phosphorylation and impair memory.

3D-QSAR and docking studies on adenosine A2A receptor antagonists by the CoMFA method.