Rapid Histamine-Induced Neutrophil Recruitment Is Sphingosine Kinase-1 Dependent

  • Wai Y. Sun
    Affiliations
    Division of Human Immunology, Centre for Cancer Biology, SA Pathology, Adelaide, Australia

    School of Medicine, University of Adelaide, Adelaide, Australia

    Co-Operative Research Centre for Biomarker Translation, La Trobe University, Bundoora, Australia
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  • Latasha D. Abeynaike
    Affiliations
    Department of Medicine, Centre for Inflammatory Diseases, Monash Medical Centre, Monash University, Clayton, Australia
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  • Samantha Escarbe
    Affiliations
    Division of Human Immunology, Centre for Cancer Biology, SA Pathology, Adelaide, Australia
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  • Charles D. Smith
    Affiliations
    Medical University of South Carolina, Charleston, South Carolina
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  • Stuart M. Pitson
    Affiliations
    Division of Human Immunology, Centre for Cancer Biology, SA Pathology, Adelaide, Australia

    Co-Operative Research Centre for Biomarker Translation, La Trobe University, Bundoora, Australia

    Molecular and Biomedical Sciences, University of Adelaide, Adelaide, Australia
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  • Michael J. Hickey
    Affiliations
    Department of Medicine, Centre for Inflammatory Diseases, Monash Medical Centre, Monash University, Clayton, Australia
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  • Claudine S. Bonder
    Correspondence
    Address reprint requests to Claudine S. Bonder, Ph.D., Centre for Cancer Biology, South Australia Pathology, Frome Road, Adelaide, SA 5000, Australia
    Affiliations
    Division of Human Immunology, Centre for Cancer Biology, SA Pathology, Adelaide, Australia

    School of Medicine, University of Adelaide, Adelaide, Australia

    Co-Operative Research Centre for Biomarker Translation, La Trobe University, Bundoora, Australia

    Molecular and Biomedical Sciences, University of Adelaide, Adelaide, Australia
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Published:February 10, 2012DOI:https://doi.org/10.1016/j.ajpath.2011.12.024
      Leukocyte recruitment to sites of inflammation is critical for the development of acute allergic responses. Rapid P-selectin up-regulation by endothelial cells is a key promoter of leukocyte infiltration in response to mediators such as histamine. However, the mechanisms underpinning this process are still incompletely understood. We examined the role of the sphingosine kinase/sphingosine-1-phosphate (SK/S1P) pathway and showed that in human umbilical vein endothelial cells, histamine rapidly activates SK in an extracellular signal-regulated kinase (ERK) 1/2-dependent manner, concurrent with the induction of P-selectin expression. Histamine activated both SK-1 and SK-2 isoforms; inhibition of SK-1, but not SK-2, attenuated histamine-induced P-selectin up-regulation and neutrophil rolling in vitro. S1P receptor antagonists failed to prevent histamine-induced P-selectin expression, and exogenous S1P did not increase P-selectin expression, suggesting that S1P cell surface receptors are not involved in this process. Finally, the role of both SK-1 and SK-2 in histamine-induced leukocyte rolling in vivo was assessed using pharmacological and genetic methods. Consistent with the in vitro findings, mice pretreated with either sphingosine kinase inhibitor or fingolimod (FTY720) significantly attenuated histamine-induced leukocyte rolling in the cremaster muscle. Similarly, Sphk1−/− but not Sphk2−/− mice exhibited reduced histamine-induced leukocyte rolling. These findings demonstrate a key role for SK-1 in histamine-induced rapid P-selectin up-regulation and associated leukocyte rolling, and suggest that endothelial SK-1 is an important contributor to allergic inflammation.
      Inflammation is central to the development of acute allergic responses. The allergic inflammatory response is a multistep process involving increased vascular permeability, changes in expression of endothelial cell adhesion molecules, and the triggering of cell-cell interactions between circulating leukocytes and the vascular endothelium. Several types of adhesion molecules are involved in leukocyte binding and transmigration, and their expression is tightly regulated to produce the sequence of events that leads to leukocyte recruitment. In allergic inflammation, these events are coordinated by inflammatory mediators, including histamine. Histamine activates the local vasculature by binding to its G-protein coupled receptors, H1 and H2, on endothelial cells and thus causing a rapid exocytosis of the preformed adhesion molecule P-selectin.
      • Burns A.R.
      • Bowden R.A.
      • Abe Y.
      • Walker D.C.
      • Simon S.I.
      • Entman M.L.
      • Smith C.W.
      P-selectin mediates neutrophil adhesion to endothelial cell borders.
      • Repka-Ramirez M.S.
      New concepts of histamine receptors and actions.
      Circulating neutrophils are immediately recruited by tethering and rolling along the vasculature via a P-selectin/P-selectin glycoprotein ligand-1 (PSGL-1) mechanism.
      • Burns A.R.
      • Bowden R.A.
      • Abe Y.
      • Walker D.C.
      • Simon S.I.
      • Entman M.L.
      • Smith C.W.
      P-selectin mediates neutrophil adhesion to endothelial cell borders.
      The ability of P-selectin to undergo a rapid increase in exposure on the endothelial surface plays a critical role in development of this initial phase of the allergic response. It is therefore important that the molecular basis of this response be completely understood.
      P-selectin is constitutively synthesized in endothelial cells,
      • Geng J.G.
      • Bevilacqua M.P.
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      • Zimmerman G.A.
      • McEver R.P.
      Rapid neutrophil adhesion to activated endothelium mediated by GMP-140.
      megakaryocytes/platelets,
      • McEver R.P.
      • Martin M.N.
      A monoclonal antibody to a membrane glycoprotein binds only to activated platelets.
      and resident peritoneal macrophages,
      • Tchernychev B.
      • Furie B.
      • Furie B.C.
      Peritoneal macrophages express both P-selectin and PSGL-1.
      where it is packaged into Weibel-Palade body and α storage granules.
      • McEver R.P.
      • Martin M.N.
      A monoclonal antibody to a membrane glycoprotein binds only to activated platelets.
      • McEver R.P.
      • Beckstead J.H.
      • Moore K.L.
      • Marshall-Carlson L.
      • Bainton D.F.
      GMP-140, a platelet alpha-granule membrane protein, is also synthesized by vascular endothelial cells and is localized in Weibel-Palade bodies.
      Two distinct mechanisms regulate the inducible expression of P-selectin. In mice, mediators such as tumor necrosis factor (TNF), interleukin-1, and lipopolysaccharide can induce transcription of P-selectin mRNA, with subsequent protein synthesis and surface expression. This response is not seen in human endothelial cells, however, because of the lack of binding sites for NF-κB and activating transcription factor-2 (ATF-2) in the human SELP gene promoter.
      • Pan J.
      • McEver R.P.
      Characterization of the promoter for the human P-selectin gene.
      • Pan J.
      • Xia L.
      • McEver R.P.
      Comparison of promoters for the murine and human P-selectin genes suggests species-specific and conserved mechanisms for transcriptional regulation in endothelial cells.
      • Yao L.
      • Setiadi H.
      • Xia L.
      • Laszik Z.
      • Taylor F.B.
      • McEver R.P.
      Divergent inducible expression of P-selectin and E-selectin in mice and primates.
      Alternatively, in both mice and humans, P-selectin can be rapidly mobilized to the endothelial surface from Weibel-Palade bodies in response to mediators such as histamine, thrombin, and other secretagogues.
      • Vestweber D.
      • Blanks J.E.
      Mechanisms that regulate the function of the selectins and their ligands [Erratum appeared in Physiol Rev 2000, 80(3):follow i].
      This mechanism does not require new protein synthesis, instead being induced by rapidly acting signaling molecules within endothelial cells. For mediators associated with allergic inflammation, such as histamine, the signaling molecules involved in this rapid response are not fully characterized, but the sphingosine kinase pathway is one candidate.
      Sphingosine kinase (SK) is a highly conserved lipid kinase. Two isoforms (SK-1 and SK-2) have been identified, cloned, and characterized.
      • Pitson S.M.
      • D'Andrea R.J.
      • Vandeleur L.
      • Moretti P.A.
      • Xia P.
      • Gamble J.R.
      • Vadas M.A.
      • Wattenberg B.W.
      Human sphingosine kinase: purification, molecular cloning and characterization of the native and recombinant enzymes.
      • Liu H.
      • Sugiura M.
      • Nava V.E.
      • Edsall L.C.
      • Kono K.
      • Poulton S.
      • Milstien S.
      • Kohama T.
      • Spiegel S.
      Molecular cloning and functional characterization of a novel mammalian sphingosine kinase type 2 isoform.
      Both SK-1 and SK-2 catalyze the phosphorylation of sphingosine to form sphingosine-1-phosphate (S1P), but they exhibit different subcellular localization patterns, developmental expression, and distribution in adult tissue and have been recognized to have both overlapping and alternative biological functions.
      • Pitson S.M.
      Regulation of sphingosine kinase and sphingolipid signaling.
      S1P is a bioactive phospholipid and is an important signaling molecule that can be either retained inside or secreted out of the cell. Basal levels of S1P in cells are generally low, but can increase rapidly when cells are exposed to various agonists through rapid and transient activation of SK activity as a result of phosphorylation on Ser225 by extracellular signal-regulated kinases 1 and 2 (ERK-1/2).
      • Pitson S.M.
      • Moretti P.A.
      • Zebol J.R.
      • Lynn H.E.
      • Xia P.
      • Vadas M.A.
      • Wattenberg B.W.
      Activation of sphingosine kinase 1 by ERK1/2–mediated phosphorylation.
      Extracellular S1P acts on its G-protein coupled receptors, S1P1–5, in both autocrine and paracrine fashions with, for example, downstream signaling of phosphatidyl inositol 3-kinase (PI3K)/Akt and ERK-1/2.
      • Pitson S.M.
      Regulation of sphingosine kinase and sphingolipid signaling.
      Alternatively, endogenous S1P can associate with histone deacetylases (HDAC1 and HDAC2),
      • Hait N.C.
      • Allegood J.
      • Maceyka M.
      • Strub G.M.
      • Harikumar K.B.
      • Singh S.K.
      • Luo C.
      • Marmorstein R.
      • Kordula T.
      • Milstien S.
      • Spiegel S.
      Regulation of histone acetylation in the nucleus by sphingosine-1-phosphate [Erratum appeared in Science 2009, 326:366].
      tumor necrosis factor receptor-associated factor 2 (TRAF2),
      • Alvarez S.E.
      • Harikumar K.B.
      • Hait N.C.
      • Allegood J.
      • Strub G.M.
      • Kim E.Y.
      • Maceyka M.
      • Jiang H.
      • Luo C.
      • Kordula T.
      • Milstien S.
      • Spiegel S.
      Sphingosine-1-phosphate is a missing cofactor for the E3 ubiquitin ligase TRAF2.
      prohibitin,
      • Strub G.M.
      • Paillard M.
      • Liang J.
      • Gomez L.
      • Allegood J.C.
      • Hait N.C.
      • Maceyka M.
      • Price M.M.
      • Chen Q.
      • Simpson D.C.
      • Kordula T.
      • Milstien S.
      • Lesnefsky E.J.
      • Spiegel S.
      Sphingosine-1-phosphate produced by sphingosine kinase 2 in mitochondria interacts with prohibitin 2 to regulate complex IV assembly and respiration.
      or as yet unidentified targets. S1P has been shown to synergize with histamine during a 4-hour exposure to promote gene and surface expression of E-selectin, ICAM-1, and VCAM-1.
      • Shimamura K.
      • Takashiro Y.
      • Akiyama N.
      • Hirabayashi T.
      • Murayama T.
      Expression of adhesion molecules by sphingosine 1-phosphate and histamine in endothelial cells.
      However, the contribution of the SK pathway to rapid leukocyte recruitment typical of allergic responses has not previously been investigated.
      With the present study, we identify SK-1 as a new potential target for controlling rapid recruitment of neutrophils after exposure to histamine. First, we demonstrate that both SK-1 and SK-2 are rapidly activated by histamine in human umbilical vein endothelial cells (HUVECs) and that this occurs in an ERK-1/2-dependent manner. Second, we demonstrate that histamine-induced surface expression of P-selectin on HUVECs requires both ERK-1/2 and SK-1 but does not involve SK-2 or the S1P1–3 surface receptors. Finally, we demonstrate that histamine-induced SK-1, but not SK-2, activity mediates neutrophil recruitment in vitro and in vivo. Collectively, the present findings suggest that SK-1 may be a critical regulator controlling acute allergic responses.

      Materials and Methods

      Reagents and Antibodies

      Antibodies against human P-selectin (AK-4) and isotype control were purchased from BD Biosciences (Franklin Lakes, NJ). Phosphorylated ERK-1/2 and total ERK-1/2 were purchased from Cell Signaling Technology (Danvers, MA). Human SK-1 antibody was generated as described previously.
      • Pitson S.M.
      • Moretti P.A.
      • Zebol J.R.
      • Lynn H.E.
      • Xia P.
      • Vadas M.A.
      • Wattenberg B.W.
      Activation of sphingosine kinase 1 by ERK1/2–mediated phosphorylation.
      Secondary antibodies anti-rabbit-HRP (Pierce; Thermo Fisher Scientific, Rockford, IL), anti-rabbit Alexa Fluor 594, anti-mouse Alexa Fluor 488, and DAPI (Invitrogen, Carlsbad, CA) were used. Human recombinant histamine, histamine-1-receptor antagonist (chlorpheniramine), and histamine-2-receptor antagonist (cimetidine) were purchased from Sigma-Aldrich (St. Louis, MO). Sphingosine kinase inhibitor (SKi) and S1P were purchased from Cayman Chemical (Ann Arbor, MI). Other inhibitors were purchased as follows: N,N-dimethyl sphingosine (DMS; Biomol International-Enzo Life Sciences, Plymouth Meeting, PA); S1P1 receptor antagonist (W146; Cayman Chemical); S1P2 receptor inhibitor (JTE013; Cayman Chemical); S1P3 receptor antagonist (CAY10444; Cayman Chemical); S1P1&3 receptor inhibitor (VPC23019; Avanti Polar Lipids, Alabaster, AL); fingolimod (FTY720; Sapphire Bioscience, Waterloo, Australia); and MAPK pathway inhibitors (U0126, Cell Signaling Technology; SB203580 and PD98059, Alexis Biochemicals-Enzo Life Sciences, Plymouth Meeting, PA). The SK-2 inhibitor ABC294640 was synthesized as described previously.
      • French K.J.
      • Zhuang Y.
      • Maines L.W.
      • Gao P.
      • Wang W.
      • Beljanski V.
      • Upson J.J.
      • Green C.L.
      • Keller S.N.
      • Smith C.D.
      Pharmacology and antitumor activity of ABC294640, a selective inhibitor of sphingosine kinase-2.

      Animals

      Wild-type (WT), SK-1 knockout (Sphk1−/−), and SK-2 knockout (Sphk2−/−) mice on a C57Bl/6 background
      • Allende M.L.
      • Sasaki T.
      • Kawai H.
      • Olivera A.
      • Mi Y.
      • van Echten-Deckert G.
      • Hajdu R.
      • Rosenbach M.
      • Keohane C.A.
      • Mandala S.
      • Spiegel S.
      • Proia R.L.
      Mice deficient in sphingosine kinase 1 are rendered lymphopenic by FTY720.
      • Kharel Y.
      • Lee S.
      • Snyder A.H.
      • Sheasley-O'neill S.L.
      • Morris M.A.
      • Setiady Y.
      • Zhu R.
      • Zigler M.A.
      • Burcin T.L.
      • Ley K.
      • Tung K.S.
      • Engelhard V.H.
      • Macdonald T.L.
      • Pearson-White S.
      • Lynch K.R.
      Sphingosine kinase 2 is required for modulation of lymphocyte traffic by FTY720.
      were housed under pathogen-free conditions at SA Pathology and at Monash University and were used between 6 and 12 weeks of age. All experimental procedures were approved by the Animal Ethics Committee of South Australia Pathology, the University of Adelaide, and Monash University and conform to the guidelines established by the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.

      Cells and Cell Culture

      The collection of human umbilical cords for use in the present study was given ethical clearance from the Human Research Ethics Committee of the Children, Youth and Women's Health Service (CYWHS), North Adelaide; informed written consent was obtained from all subjects in accordance with the Declaration of Helsinki. Human umbilical vein endothelial cells (HUVECs) were isolated as described previously.
      • Litwin M.
      • Clark K.
      • Noack L.
      • Furze J.
      • Berndt M.
      • Albelda S.
      • Vadas M.
      • Gamble J.
      Novel cytokine-independent induction of endothelial adhesion molecules regulated by platelet/endothelial cell adhesion molecule (CD31).
      HUVECs were grown in M199 medium (Sigma-Aldrich) containing 20% human serum (Invitrogen), 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen, Gibco BRL, Paisley, Scotland). Cells were cultured on 10% gelatin (Sigma-Aldrich) and used at passage 1.
      Neutrophils and lymphocytes were enriched from venipuncture samples of consenting healthy donors, as described previously.
      • Eggleton P.
      • Gargan R.
      • Fisher D.
      Rapid method for the isolation of neutrophils in high yield without the use of dextran or density gradient polymers.
      Briefly, after dextran sedimentation the cells were enriched by density-gradient centrifugation on Lymphoprep medium (Nycomed, Oslo, Norway), with the neutrophils pelleting at the base and the lymphocytes enriched at the interface. After hypotonic lysis of erythrocytes, cells were resuspended in RPMI 1640 medium containing 10 mmol/L HEPES and 2.5% fetal bovine serum (Invitrogen, Gibco BRL) before use. Cytological examination of cytocentrifuged preparations with May-Grünwald Giemsa staining (Sigma-Aldrich) showed that >95% of the cells were neutrophils or lymphocytes. Trypan Blue staining confirmed that >98% of these cells were viable. The human Jurkat T-cell line was cultured in complete RPMI 1640 medium (Gibco BRL) with 10% fetal bovine serum. To quantify the degree of Jurkat cell activation in response to histamine (25 μmol/L, 30 minutes) or phorbol myristate acetate (100 ng/mL 30 minutes), levels of L-selectin expression were measured using flow cytometry with 1 μg of monoclonal antibody against L-selectin (Dreg56 mouse anti-human, a kind gift from E. Butcher) or a nonspecific isotype control (IgG1; BD Biosciences) for 30 minutes on ice. Cells were then washed and incubated with Alexa Fluor 488-conjugated anti-mouse Ig (1:1000 dilution; Invitrogen) for 30 minutes on ice. Stained cells were resuspended in fluorescence-activated cell sorting Fix medium (1% formaldehyde, 20 g/L glucose, 5 mmol/L sodium azide in PBS) before analysis using a Beckman Coulter XL-MCL using CXP Cytometry List Mode Data Acquisition & Analysis Software version 2.2 (Gladesville, Australia). Further analysis was performed using FCS Express version 3.0 software (De Novo Software, Los Angeles, CA) against unstained cells gated at ≤1%.

      SK Activity Assay

      SK activity was determined as described previously.
      • Pitson S.M.
      • D'Andrea R.J.
      • Vandeleur L.
      • Moretti P.A.
      • Xia P.
      • Gamble J.R.
      • Vadas M.A.
      • Wattenberg B.W.
      Human sphingosine kinase: purification, molecular cloning and characterization of the native and recombinant enzymes.
      For SK-1 activity, whole-cell lysates were incubated with d-erythro sphingosine (Biomol) solubilized in either 0.05% or 0.1% Triton X-100 and [γ32P]ATP (PerkinElmer, Melbourne, Australia). For SK-2 activity, whole-cell lysates were prepared in buffer containing 1 mol/L KCl and incubated with d-erythro sphingosine solubilized in bovine serum albumin/PBS and [γ32P]ATP. The radiolabeled S1P was resolved by two thin-layer chromatography (Sigma-Aldrich) separations in the solvents containing butanol, ethanol, water, and acetic acid (8:2:2:1). The radioactive spots were quantified using Phosphorimaging Typhoon 9410 (Beckman Coulter, Fullerton, CA) and ImageQuant software version 5.2 (GE Healthcare, Rydalmere, Australia).

      Western Blotting

      HUVECs were lysed in buffer containing 1% NP40 surfactant and then sonicated. Cell lysates were separated by 10% SDS-PAGE and transferred to Hybond-P membrane (Amersham; GE Healthcare, Piscataway, NJ). Primary antibodies to pERK-1/2 or total ERK-1/2 were used to probe the membrane overnight at 4°C, followed by secondary antibody incubation at room temperature (RT) for 1 hour before visualization by enzymatic chemiluminescence (GE Healthcare) and a luminescent image analyzer (LAS4000; Fujifilm, Stamford, CT).

      MAPK, SK, and S1P-Receptor Inhibition and S1P-Receptor Activation Studies

      In the activation and inhibition studies, SK inhibitor (SKi; 5 μmol/L, 10 minutes), DMS (5 μmol/L, 10 minutes), ERK-1/2 pathway inhibitor (U0126; 10 μmol/L, 30 minutes), p38 inhibitor (SB203580; 10 μmol/L, 1 hour), MEK inhibitor (PD98059; 25 μmol/L, 30 minutes), S1P (1 μmol/L, 10 minutes), fingolimod (FTY720, 100 nmol/L, 30 minutes), JTE013 (1 μmol/L, 30 minutes), W146 (10 μmol/L, 30 minutes), CAY10444 (10 μmol/L, 30 minutes), or VPC23019 (10 μmol/L, 30 minutes) were administered before histamine stimulation (25 μmol/L, 5 minutes). All reagents were proven functionally effective in paralleled studies.

      Immunofluorescence Microscopy

      HUVECs were replated at 5 × 104 cells/well in fibronectin-coated (50 μg/mL) Lab-Tek chamber slides (Nalge Nunc International, Rochester, NY). Confluent cells were treated with SKi, DMS, S1P, JTE013, VPC23019, W146, CAY10444, fingolimod, U0126, SB203580, PD98059, chlorpheniramine, or cimetidine without or with histamine stimulation (25 μmol/L, 5 minutes). Cells were fixed with 4% paraformaldehyde at RT for 15 minutes before blocking with 2% bovine serum albumin/PBS at RT for 30 minutes. P-selectin antibody (1 μg/mL) was added to cells overnight at 4°C, followed by anti-rabbit Alexa Fluor 594-conjugated antibody (1:1000) incubation at RT for 1 hour. Cells were then permeabilized with 0.1% Triton-X 100/PBS at RT for 10 minutes, followed by DAPI staining (1:2000) at RT for 3 minutes. Slides were visualized under an Olympus IX70 inverted microscope (Olympus, Tokyo, Japan) linked to a Bio-Rad Radiance 2100 confocal microscope (Bio-Rad Laboratories, Hercules, CA; Gladesville, Australia). Five images were acquired per sample. The fluorescence intensity was analyzed using Olympus AnalySIS Life Science imaging software version 3.0.

      Parallel Plate Flow Chamber Assay

      Confluent HUVECs cultured on Corning Petri dishes (Sigma-Aldrich) were treated with isotype control antibody (10 μg/mL, 30 minutes), P-selectin blocking antibody (10 μg/mL, 30 minutes), SKi (5 μmol/L, 10 minutes), DMS (5 μmol/L, 10 minutes), fingolimod (100 nmol/L, 30 minutes), ABC294640 (10 μmol/L, 10 minutes), U0126 (10 μmol/L, 30 minutes), PD98059 (25 μmol/L, 30 minutes), or SB203580 (10 μmol/L, 1 hour) before perfusion of histamine (25 μmol/L, 2.5 minutes) followed by blood, neutrophils, or lymphocytes. Using published methods, histamine (25 μmol/L) was prepared in Hank's balanced salt solution (HBSS; Sigma-Aldrich) and perfused across the substratum using a syringe pump (NE-1000; New Era Pump Systems, Farmingdale, NY) at a constant rate of 2 dynes/cm2 for 2.5 minutes.
      • Bonder C.S.
      • Clark S.R.
      • Norman M.U.
      • Johnson P.
      • Kubes P.
      Use of CD44 by CD4+ Th1 and Th2 lymphocytes to roll and adhere.
      Peripheral blood was obtained by venipuncture from healthy donors after informed consent into heparinized syringes; samples were diluted 1:10 with HBSS and then perfused for 5 minutes, followed by an HBSS wash. Alternatively, blood in acid-citrate-dextrose was used to isolate neutrophils or lymphocytes before perfusion at 1 × 106 cells/mL for 5 minutes, followed by HBSS wash. Unlabeled leukocyte, neutrophil, or lymphocyte interactions were visualized under phase-contrast microscopy using 10×/0.3 NA objectives on an inverted microscope. Five random areas per dish were recorded using a digital camera (Olympus IX70 and SIS F-view) and analyzed using AnalySIS Life Science imaging software version 3.0 (Olympus). The number of rolling cells was analyzed using the aforementioned video microscopy system.

      Intravital Microscopy and in Vivo Experimental Procedure

      Intravital microscopy of the cremaster muscle was performed as described previously.
      • Gregory J.L.
      • Leech M.T.
      • David J.R.
      • Yang Y.H.
      • Dacumos A.
      • Hickey M.J.
      Reduced leukocyte-endothelial cell interactions in the inflamed microcirculation of macrophage migration inhibitory factor-deficient mice.
      Briefly, microscopy (Axioplan 2 Imaging; Carl Zeiss Australia, Carnegie, Australia) with a 20× objective lens (20×/0.40 numerical aperture) and 10× eyepiece was used to observe the cremasteric microcirculation. A color video camera (Sony SSC-DC50AP; Carl Zeiss Australia) was used to project the images onto a calibrated monitor (Sony PVM-20N5E), and the images were recorded for playback analysis using a DVD recorder (Panasonic DMR-EH57; Retravision, Moorabbin, Australia). Two postcapillary venules (25 to 40 μm in diameter) were examined for each experiment. Leukocyte rolling was assessed via playback analysis, as described previously.
      • Gregory J.L.
      • Leech M.T.
      • David J.R.
      • Yang Y.H.
      • Dacumos A.
      • Hickey M.J.
      Reduced leukocyte-endothelial cell interactions in the inflamed microcirculation of macrophage migration inhibitory factor-deficient mice.
      In experiments examining the effect of SK inhibition, WT mice were injected subcutaneously with vehicle alone or SKi (50 mg/kg in dimethyl sulfoxide/PBS) for 15 minutes or injected intraperitoneally with fingolimod (0.5 mg/kg in PBS) for 60 minutes before intravital microscopy. A basal reading of leukocyte rolling flux was taken before histamine superfusion (100 μmol/L in superfusion buffer) commenced. Additional recordings of leukocyte rolling were subsequently made at 5, 10, 20, and 30 minutes after histamine superfusion commenced. In a separate series of experiments, WT, Sphk1−/−, and Sphk2−/− mice underwent the same model of histamine challenge.

      Statistical Analysis

      Data were statistically analyzed by Student's t-test or one-way or two-way analysis of variance for multiple comparisons and are expressed as means ± SEM. P < 0.05 was considered significant.

      Results

      Histamine Rapidly Induces P-Selectin Expression and SK Activity in HUVECs

      On activation by histamine, the vascular endothelium rapidly expresses preformed P-selectin at the cell surface for an immediate inflammatory response of leukocyte recruitment from the circulation and rolling along the vasculature.
      • Johnson R.C.
      • Mayadas T.N.
      • Frenette P.S.
      • Mebius R.E.
      • Subramaniam M.
      • Lacasce A.
      • Hynes R.O.
      • Wagner D.D.
      Blood cell dynamics in P-selectin-deficient mice.
      In the present study, we used immunofluorescence microscopy to demonstrate that exposure of HUVECs to histamine for 5 minutes rapidly induces the surface expression of P-selectin (Figure 1A). Histamine-induced P-selectin surface expression is not associated with increased mRNA levels (data not shown) and occurs via the H1 receptor, because pretreatment of HUVECs with the H1 receptor antagonist chlorpheniramine but not the H2 receptor antagonist cimetidine inhibited these events (Figure 1B).
      • Molet S.
      • Gosset P.
      • Lassalle P.
      • Czarlewski W.
      • Tonnel A.B.
      Inhibitory activity of loratadine and descarboxyethoxyloratadine on histamine-induced activation of endothelial cells.
      Figure thumbnail gr1
      Figure 1Histamine rapidly promotes P-selectin expression via the H1 receptor and activates SK-1 and SK-2. A: Immunofluorescence microscopy of HUVECs treated for 5 minutes without or with 25 μmol/L histamine or 5 ng/mL TNF before P-selectin staining (red), permeabilization, and DAPI staining (blue). A representative image is shown (n = 3). NT, no treatment. Original magnification, ×100. B: Pooled fluorescence intensity data of histamine-treated HUVECs without and with H1 antagonist (chlorpheniramine) or H2 antagonist (cimetidine). Data are expressed as means ± SEM (n = 3). *P < 0.05 versus untreated. C: HUVECs stimulated without and with 25 μmol/L histamine for 2.5, 5, 10, and 30 minutes before lysis for SK enzymatic assay. Data are expressed as means ± SEM (n = 6). D and E: HUVECs were preincubated with either SK-1 inhibitor (SKi; 5 μmol/L) or SK-2 inhibitor (ABC294640; 10 μmol/L) 10 minutes before histamine stimulation for 5 minutes. Cells were lysed immediately for SK-1 (D) or SK-2 (E) enzymatic assay. Data are expressed as means ± SEM (n = 3 to 6). *P < 0.05 versus untreated; P < 0.05 versus histamine.
      Huwiler et al
      • Huwiler A.
      • Döll F.
      • Ren S.
      • Klawitter S.
      • Greening A.
      • Römer I.
      • Bubnova S.
      • Reinsberg L.
      • Pfeilschifter J.
      Histamine increases sphingosine kinase-1 expression and activity in the human arterial endothelial cell line EA.hy 926 by a PKC-alpha-dependent mechanism.
      demonstrated that prolonged exposure to histamine (>2 hours) increases SK-1 expression and activity in a human arterial endothelial cell line, and we recently demonstrated that TNFα-induced SK activity in HUVECs occurs in a biphasic manner, with peaks observed both at 10 minutes and at 4 to 6 hours after treatment.
      • Sun W.Y.
      • Pitson S.M.
      • Bonder C.S.
      Tumor necrosis factor-induced neutrophil adhesion occurs via sphingosine kinase-1-dependent activation of endothelial {alpha}5{beta}1 integrin.
      Based on these observations, we hypothesized that histamine activates SK within minutes of exposure. Indeed, this appears to be the case. A time-course treatment of 25 μmol/L histamine on HUVECs demonstrated an increase in SK activity at 2.5 minutes, peaking at 10 minutes and subsiding at 30 minutes (Figure 1C). Because TNF is also known to increase SK activity in HUVECs within minutes,
      • Sun W.Y.
      • Pitson S.M.
      • Bonder C.S.
      Tumor necrosis factor-induced neutrophil adhesion occurs via sphingosine kinase-1-dependent activation of endothelial {alpha}5{beta}1 integrin.
      we investigated whether TNF could also exocytose P-selectin to the cell surface. The commonality observed between histamine and TNF in rapidly activating SK in HUVECs does not seem to extend to P-selectin exocytosis on these cells (Figure 1A).
      To investigate whether the SK-1 or SK-2 isoform is preferentially activated by histamine, we executed experiments wherein the addition of 0.1% Triton X-100 or 1 mol/L KCl in the enzymatic assay is used to distinguish between SK-1 and SK-2 activity, respectively.
      • Liu H.
      • Sugiura M.
      • Nava V.E.
      • Edsall L.C.
      • Kono K.
      • Poulton S.
      • Milstien S.
      • Kohama T.
      • Spiegel S.
      Molecular cloning and functional characterization of a novel mammalian sphingosine kinase type 2 isoform.
      HUVECs exposed to histamine for 5 minutes exhibited increased activity of both SK-1 and SK-2, with SK-1 activity approximately twofold higher than that of SK-2 (Figure 1, D and E). Notably, unstimulated HUVECs exhibited equivalent levels of basal SK-1 and SK-2 activity (data not shown). The specificity of these assays was confirmed in experiments using HUVECs pretreated with SKi
      • French K.J.
      • Schrecengost R.S.
      • Lee B.D.
      • Zhuang Y.
      • Smith S.N.
      • Eberly J.L.
      • Yun J.K.
      • Smith C.D.
      Discovery and evaluation of inhibitors of human sphingosine kinase.
      and the SK-2 inhibitor ABC294640,
      • French K.J.
      • Zhuang Y.
      • Maines L.W.
      • Gao P.
      • Wang W.
      • Beljanski V.
      • Upson J.J.
      • Green C.L.
      • Keller S.N.
      • Smith C.D.
      Pharmacology and antitumor activity of ABC294640, a selective inhibitor of sphingosine kinase-2.
      • Ren S.
      • Xin C.
      • Pfeilschifter J.
      • Huwiler A.
      A novel mode of action of the putative sphingosine kinase inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (SKI II): induction of lysosomal sphingosine kinase 1 degradation.
      which demonstrated selective reductions in activity of the two SK isoforms (Figure 1, D and E).

      Histamine-Induced SK Activity in HUVECs is ERK-1/2 dependent

      The catalytic activity of SK can be rapidly and transiently activated by a diverse range of growth factors, cytokines, and other cell agonists
      • Pitson S.M.
      Regulation of sphingosine kinase and sphingolipid signaling.
      via phosphorylation on Ser225 by ERK-1/2.
      • Pitson S.M.
      • Moretti P.A.
      • Zebol J.R.
      • Lynn H.E.
      • Xia P.
      • Vadas M.A.
      • Wattenberg B.W.
      Activation of sphingosine kinase 1 by ERK1/2–mediated phosphorylation.
      We next investigated whether the signaling pathways by which histamine activates SKs in endothelial cells also involve the phosphorylation of ERK-1/2. The 25 μmol/L histamine treatment significantly increased the phosphorylation of ERK-1/2 at 5 minutes (Figure 2, A and B); phosphorylation peaked at 10 minutes and subsided at 20 minutes after exposure. Notably, the timing of ERK-1/2 phosphorylation parallels that observed for histamine-induced SK activity (Figure 1D). Blocking the ERK-1/2 pathway by administration of U0126 prevented histamine-induced SK activity in HUVECs (Figure 2C). Inhibition of SK by SKi had no effect on histamine-induced ERK-1/2 phosphorylation (Figure 2D), consistent with ERK-1/2 activation being upstream of SK activity. As expected, SK-1 protein levels did not alter during short-term exposure of HUVECs to 25 μmol/L histamine (see Supplemental Figure S1 at http://ajp.amjpathol.org).
      Figure thumbnail gr2
      Figure 2Histamine increases phosphorylation of ERK-1/2, which activates SK. A: HUVECs were treated without and with histamine (25 μmol/L for 5, 10, and 20 minutes) before lysis and Western blotting for phosphorylated ERK-1/2 (pERK) and total ERK-1/2 (ERK). Representative blots are shown (n = 4). B: Pooled data are expressed as means ± SEM (n = 4). *P < 0.05 versus untreated. C: HUVECs were pretreated with ERK-1/2 pathway inhibitor (U0126; 10 μmol/L, 30 minutes) before histamine stimulation (25 μmol/L, 5 minutes) and lysis for SK enzymatic assay. Data are expressed as means ± SEM (n = 5 to 7). *P < 0.05 versus untreated; P < 0.05 versus histamine. D: HUVECs were pretreated with SKi (5 μmol/L, 10 minutes) before histamine stimulation (25 μmol/L, 5 minutes) and examined for phosphorylated and total ERK-1/2 by Western blotting. Data are expressed as means ± SEM (n = 5). *P < 0.05 versus untreated.

      Histamine-Induced P-selectin Surface Expression Is ERK-1/2 and SK-1 Dependent but Is S1P Surface Receptor Independent

      Using immunofluorescence microscopy, we next examined a direct link between the MAPK pathway, SKs, and P-selectin surface expression on histamine-treated HUVECs. First, HUVECs treated with the ERK-1/2 pathway inhibitor U0126 before histamine administration exhibited a reduction in P-selectin surface expression similar to that observed in the absence of histamine (Figure 3A). A similar reduction in histamine-induced P-selectin expression was observed with administration of the MEK inhibitor PD98059 but not the p38 inhibitor SB203580 (Figure 3A). Second, two separate SK inhibitors [dimethyl sphingosine (DMS), a competitive inhibitor for both SK-1 and SK-2,
      • Pitson S.M.
      • D'Andrea R.J.
      • Vandeleur L.
      • Moretti P.A.
      • Xia P.
      • Gamble J.R.
      • Vadas M.A.
      • Wattenberg B.W.
      Human sphingosine kinase: purification, molecular cloning and characterization of the native and recombinant enzymes.
      • Liu H.
      • Sugiura M.
      • Nava V.E.
      • Edsall L.C.
      • Kono K.
      • Poulton S.
      • Milstien S.
      • Kohama T.
      • Spiegel S.
      Molecular cloning and functional characterization of a novel mammalian sphingosine kinase type 2 isoform.
      and SKi] were used to examine the role of SK in histamine-induced P-selectin expression. A significant reduction in histamine-induced P-selectin expression was observed when HUVECs were pretreated with either DMS or SKi (Figure 3A). These results suggest that histamine-induced P-selectin expression is SK dependent.
      Figure thumbnail gr3
      Figure 3Inhibition of ERK-1/2 pathway or SK attenuates histamine-induced P-selectin surface expression in an S1P1–3 receptor-independent manner. A: HUVECs were preincubated with U0126 (10 μmol/L, 30 minutes), PD98059 (25 μmol/L, 30 minutes), DMS (5 μmol/L, 10 minutes), SKi (5 μmol/L, 10 minutes), SB203580 (10 μmol/L, 1 hour) without or with histamine treatment (25 μmol/L, 5 minutes) and examined for P-selectin surface expression under immunofluorescence microscopy. B: HUVECs were treated with S1P1 inhibitor (W146; 10 μmol/L, 30 minutes), S1P2 inhibitor (JTE013; 1 μmol/L, 30 minutes), S1P3 inhibitor (CAY10444; 10 μmol/L, 30 minutes), S1P1&3 inhibitor (VPC23019; 10 μmol/L, 30 minutes), or fingolimod (100 nmol/L, 30 minutes) before histamine exposure (25 μmol/L, 5 minutes). Similarly, exogenous S1P (1 μmol/L, 30 minutes) was added to HUVECs. Cells were fixed and assessed for P-selectin expression under immunofluorescence microscopy. Data are expressed as means ± SEM for quantified fluorescence intensity (n = 3 or 4). *P < 0.05 versus corresponding untreated; P < 0.05 versus histamine.
      Given that S1P1–2 receptors are known regulators of mast-cell function during an allergic response,
      • Jolly P.S.
      • Bektas M.
      • Olivera A.
      • Gonzalez-Espinosa C.
      • Proia R.L.
      • Rivera J.
      • Milstien S.
      • Spiegel S.
      Transactivation of sphingosine-1-phosphate receptors by FcepsilonRI triggering is required for normal mast cell degranulation and chemotaxis.
      and that S1P1–3 proteins have been identified on the surface of HUVECs,
      • Lin C.I.
      • Chen C.N.
      • Lin P.W.
      • Lee H.
      Sphingosine 1-phosphate regulates inflammation-related genes in human endothelial cells through S1P1 and S1P3.
      we used inhibitors for these three family members (W146 for S1P1, JTE013 for S1P2, CAY10444 for S1P3, and VPC23019 for S1P1&3) to investigate whether S1P receptors are involved in histamine-induced P-selectin expression on endothelial cells. Histamine-treated HUVECs exhibited a significant increase in P-selectin expression that was not affected by administration of inhibitors to S1P1–3 (Figure 3B). Notably, blocking S1P1, S1P3, or S1P1&3 reduced histamine-induced P-selectin expression by approximately 30%, but expression was still significantly greater than that of untreated controls (Figure 3B). To further evaluate whether the S1P receptors are involved, 1 μmol/L exogenous S1P was added to HUVECs, a concentration thought to engage only the receptors for signaling events.
      • Olivera A.
      • Spiegel S.
      Sphingosine kinase: a mediator of vital cellular functions.
      S1P treatment of HUVECs did not induce P-selectin expression (Figure 3B). Collectively, these findings suggest that S1P1–3 receptors play no major role in histamine-induced P-selectin expression by HUVECs. Also of interest, we investigated the effect of fingolimod, a sphingosine-like fungal metabolite with demonstrated direct inhibition of SK-1.
      • Vessey D.A.
      • Kelley M.
      • Zhang J.
      • Li L.
      • Tao R.
      • Karliner J.S.
      Dimethylsphingosine and FTY720 inhibit the SK1 form but activate the SK2 form of sphingosine kinase from rat heart.
      • Lee W.J.
      • Yoo H.S.
      • Suh P.G.
      • Oh S.
      • Lim J.S.
      • Lee Y.M.
      Sphingosine mediates FTY720-induced apoptosis in LLC-PK1 cells.
      • Tonelli F.
      • Lim K.G.
      • Loveridge C.
      • Long J.
      • Pitson S.M.
      • Tigyi G.
      • Bittman R.
      • Pyne S.
      • Pyne N.J.
      FTY720 and (S)-FTY720 vinylphosphonate inhibit sphingosine kinase 1 and promote its proteasomal degradation in human pulmonary artery smooth muscle, breast cancer and androgen-independent prostate cancer cells.
      Pretreatment of HUVECs with fingolimod significantly reduced histamine-induced P-selectin expression (Figure 3B).

      Leukocyte Rolling on Histamine-Treated HUVECs Is SK-1 Dependent

      We next examined the role for the MAPK pathway, SK, and P-selectin in histamine-induced recruitment of leukocytes in vitro by a parallel plate flow chamber assay. When human blood was perfused over untreated HUVECs at a physiological constant shear rate of 2 dynes/cm2, very few leukocytes rolled along the endothelium (Figure 4A). In contrast, HUVECs preperfused with 25 μmol/L histamine for 2.5 minutes demonstrated a profound increase in the number of rolling leukocytes, with approximately 100 cells per field of view (FOV). Adhesion of leukocytes was minimal to nonexistent on both untreated and histamine-treated cells (data not shown). Administration of a blocking antibody to P-selectin (AK-4) for 30 minutes before flow chamber assay significantly reduced the number of rolling leukocytes (Figure 4A).
      Figure thumbnail gr4
      Figure 4Histamine-induced leukocyte and neutrophil rolling in vitro is ERK-1/2 and SK-1 dependent but independent of S1P1–3 surface receptors. A: HUVECs were preincubated without or with an isotype control antibody (10 μg, 30 minutes), P-selectin blocking antibody (10 μg, 30 minutes), ERK-1/2 inhibitor (U0126; 10 μmol/L, 30 minutes), MEK inhibitor (PD98059; 25 μmol/L, 30 minutes), SK inhibitors (SKi or DMS; each at 5 μmol/L, 10 minutes), p38 inhibitor (SB203580; 10 μmol/L, 1 hour), SK-2 inhibitor (ABC294640; 10 μmol/L, 10 minutes), or fingolimod (100 nmol/L, 30 minutes) before perfusion of histamine (25 μmol/L, 2.5 minutes) and then human whole blood (5 minutes). Data are expressed as means ± SEM for rolling cells per FOV, with four to five FOV captured (n = 3 or 4). *P < 0.05 versus untreated; P < 0.05 versus histamine. B: HUVECs were perfused without or with histamine (25 μmol/L, 2.5 minutes) and freshly isolated human neutrophils or lymphocytes at 1 × 106 cells/mL. Data are expressed as means ± SEM for rolling flux per FOV, with four to five FOV captured (n = 3). *P < 0.05 versus untreated. C: HUVECs were pretreated without or with a control antibody (10 μg, 30 minutes), P-selectin blocking antibody (10 μg, 30 minutes), SK-1 inhibitor (SKi; 5 μmol/L, 10 minutes), or SK-2 inhibitor (ABC294640; 10 μmol/L, 10 minutes) before perfusion of histamine (25 μmol/L, 2.5 minutes) and freshly isolated human neutrophils at 1 × 106 cells/mL. Data are expressed as means ± SEM for rolling flux per FOV, with four to five FOV captured (n = 3 to 5). *P < 0.05 versus untreated; P < 0.05 versus histamine.
      For investigation of a role for ERK-1/2 and SK-1 in this system, specific inhibitors were added before histamine perfusion. A reduction in leukocyte rolling was observed when inhibitors to either the ERK pathway (U0126 and PD98059) or the SK pathway (DMS and SKi) were added (Figure 4A; see also Supplemental Video S1 at http://ajp.amjpathol.org). No reduction was observed with inhibition of the p38 pathway (SB203580) or with administration of the SK-2 inhibitor ABC294640 (Figure 4A). Consistent with our P-selectin expression data, short-term exposure of HUVECs to S1P failed to activate leukocyte rolling (data not shown). This supports the observations of histamine-induced P-selectin expression being S1P1–3 receptor independent. Of note, pretreatment with fingolimod also significantly suppressed histamine-induced leukocyte rolling (Figure 4A), suggesting a potential utility for fingolimod in the early phase of allergic inflammation.
      Because the leukocyte rolling studies to this point were performed with whole blood, we next asked whether these responses were also seen using isolated lymphocytes and neutrophils; for the latter, rolling capabilities on histamine-activated endothelial cells have been demonstrated.
      • Lorant D.E.
      • Patel K.D.
      • McIntyre T.M.
      • McEver R.P.
      • Prescott S.M.
      • Zimmerman G.A.
      Coexpression of GMP-140 and PAF by endothelium stimulated by histamine or thrombin: a juxtacrine system for adhesion and activation of neutrophils.
      Although very few, if any, lymphocytes exhibited rolling events, approximately 75 neutrophils rolled per FOV on histamine-treated HUVECs (Figure 4B). Because the lymphocytes isolated from peripheral blood are likely naïve rather than memory or effector T cells, we used histamine to preactivate Jurkat T cells and investigated their ability to interact with HUVECs. L-selectin shedding was observed on histamine-treated Jurkat cells (see Supplemental Figure S2, A and B, at http://ajp.amjpathol.org), thereby confirming an active state; however, this does not correlate with increased rolling on histamine-treated HUVECs. Blocking P-selectin by antibody administration significantly attenuated the neutrophil rolling events (Figure 4C). Similarly, HUVECs pretreated with SKi demonstrated reduced neutrophil rolling (Figure 4C). This was not observed with the SK-2 inhibitor ABC294640 (Figure 4C). Collectively, these findings suggest that histamine-induced neutrophil recruitment occurs via an SK-1-mediated P-selectin dependent process.

      SK-1 Mediates Histamine-Induced Leukocyte Rolling in Vivo

      We next performed in vivo experiments using intravital microscopy to assess the role for SKs in histamine-induced leukocyte rolling in cremasteric postcapillary venules. First, leukocyte rolling was assessed in WT mice pretreated with either SKi or vehicle. In vehicle-treated mice, histamine exposure rapidly increased leukocyte rolling flux from a basal level of ∼50 cells/minute to a peak of 168 ± 28 cells/minute within 5 minutes, before rapidly returning to basal levels (Figure 5A; see also Supplemental Video S2 at http://ajp.amjpathol.org). These mice also exhibited a transient reduction in rolling velocity from 89 ± 6 μmol/L per second to 41 ± 7 μmol/L per second, which previous studies have shown is associated with increased sensitivity to chemoattractants.
      • Kanwar S.
      • Johnston B.
      • Kubes P.
      Leukotriene C4/D4 induces P-selectin and sialyl Lewis(x)-dependent alterations in leukocyte kinetics in vivo.
      • Hickey M.J.
      • Kanwar S.
      • McCafferty D.M.
      • Granger D.N.
      • Eppihimer M.J.
      • Kubes P.
      Varying roles of E-selectin and P-selectin in different microvascular beds in response to antigen.
      As expected from the in vitro studies, mice injected subcutaneously with SKi exhibited a significantly lower peak rolling flux (89 ± 28 cells/minute) at the same time point (Figure 5A; see also Supplemental Video S2 at http://ajp.amjpathol.org), supporting the concept that histamine-induced leukocyte rolling in vivo is SK-1 dependent. Treatment of Sphk1−/− mice with SKi caused no further reduction in rolling, consistent with this agent being specific for SK-1 (data not shown). Administration of fingolimod 60 minutes before histamine exposure also significantly attenuated neutrophil rolling in vivo (Figure 5A; see also Supplemental Video S2 at http://ajp.amjpathol.org). Notably, the residual rolling neutrophils in the fingolimod-treated mice did not exhibit a reduced rolling velocity (73 ± 11 μmol/L per second versus 67 ± 17 μmol/L per second).
      Figure thumbnail gr5
      Figure 5Histamine-induced neutrophil rolling flux in response to SK inhibitors and Sphk knockout in mice. A: WT mice were injected with vehicle dimethyl sulfoxide/PBS (squares), SKi (50 mg/kg, triangles) subcutaneously 15 minutes, or fingolimod (0.5 mg/kg, circles) i.p. 60 minutes before histamine challenge (100 μmol/L, superfused topically over the cremaster muscle) and were examined under intravital microscopy. B: Similarly, WT (squares), Sphk1−/− (circles), and Sphk2−/− (triangles) mice were superfused with histamine. Leukocyte rolling flux in the postcapillary venules of the mouse cremaster muscle was assessed at 5, 10, 20, and 30 minutes after histamine challenge. Data are expressed as means ± SEM (n = 5 to 7 mice per group). *P < 0.05 versus WT.
      Second, to investigate the respective roles of SK-1 and SK-2 in histamine-induced leukocyte rolling in vivo, we used Sphk1−/− and Sphk2−/− mice. Except that the experiments performed in the Sphk1−/− mice were in postcapillary venules of a slightly reduced diameter, equivalent vascular parameters, hemodynamic parameters, and systemic leukocyte counts were observed both in the present study (Table 1) and as reported by others.
      • Allende M.L.
      • Sasaki T.
      • Kawai H.
      • Olivera A.
      • Mi Y.
      • van Echten-Deckert G.
      • Hajdu R.
      • Rosenbach M.
      • Keohane C.A.
      • Mandala S.
      • Spiegel S.
      • Proia R.L.
      Mice deficient in sphingosine kinase 1 are rendered lymphopenic by FTY720.
      • Kharel Y.
      • Lee S.
      • Snyder A.H.
      • Sheasley-O'neill S.L.
      • Morris M.A.
      • Setiady Y.
      • Zhu R.
      • Zigler M.A.
      • Burcin T.L.
      • Ley K.
      • Tung K.S.
      • Engelhard V.H.
      • Macdonald T.L.
      • Pearson-White S.
      • Lynch K.R.
      Sphingosine kinase 2 is required for modulation of lymphocyte traffic by FTY720.
      Furthermore, the equivalent baseline level of neutrophil rolling in the WT, Sphk1−/−, and Sphk2−/− mice is indicative of constitutive P-selectin expression in the cremasteric microvasculature of these strains.
      • Hickey M.J.
      • Kanwar S.
      • McCafferty D.M.
      • Granger D.N.
      • Eppihimer M.J.
      • Kubes P.
      Varying roles of E-selectin and P-selectin in different microvascular beds in response to antigen.
      • Eppihimer M.J.
      • Wolitzky B.
      • Anderson D.C.
      • Labow M.A.
      • Granger D.N.
      Heterogeneity of expression of E- and P-selectins in vivo.
      The WT mice exhibited a peak rolling flux of 142 ± 12 cells/minute after 5 minutes of histamine superfusion (Figure 5B; see also Supplemental Video S3, available at http://ajp.amjpathol.org). In Sphk2−/− mice, a slight but nonsignificant decrease in peak rolling flux (109 ± 12 cells/minute) was observed. In contrast, Sphk1−/− mice demonstrated a profound reduction in histamine-induced rolling (63 ± 11 cells/minute), supporting our in vitro data of SK-1 being the dominant SK isoform mediating histamine-induced neutrophil rolling.
      Table 1Hemodynamic State of Untreated Animals
      VariableWTSphK1−/−SphK2−/−
      Vascular diameter (μm)32.3 ± 1.028.4 ± 1.7
      P < 0.05 versus WT (n = 5 to 16).
      29.9 ± 1.9
      Mean red blood cell velocity (mm/second)2.1 ± 0.61.1 ± 0.22.3 ± 0.4
      Shear rate (s−1)463 ± 126318 ± 39616 ± 83
      Leukocyte count (no.)
       Lymphocytes65 ± 466 ± 263 ± 2
       Neutrophils28 ± 524 ± 425 ± 2
       Monocytes7 ± 310 ± 112 ± 1
      low asterisk P < 0.05 versus WT (n = 5 to 16).

      Discussion

      Investigation of the cellular and soluble mediators involved in allergic inflammation not only contributes to understanding of the mechanisms of current treatments, but is also important for the identification of new targets. With the present study, we demonstrate for the first time that SK-1 mediates the early phase of histamine-induced P-selectin-mediated neutrophil recruitment. Evidence for this comes from experiments showing that i) histamine increased ERK-1/2 phosphorylation and SK activity in HUVECs; ii) inhibition of either the ERK-1/2 pathway or SK-1, but not SK-2, markedly attenuated histamine-induced P-selectin surface expression on endothelial cells; iii) addition of S1P or inhibition of S1P1–3 receptors on histamine-treated HUVECs did not alter P-selectin surface expression; iv) histamine-induced neutrophil rolling on endothelium in vitro was P-selectin and SK-1 dependent; and v) histamine-induced neutrophil influx in vivo was significantly reduced in WT mice pretreated with an SK-1 inhibitor, as well as in Sphk1−/− mice, compared with the WT and Sphk2−/− counterparts.
      The importance of P-selectin in allergic inflammation has been well described, with an in vivo study showing that P-selectin deficient mice exhibit a significant reduction in leukocyte rolling,
      • Johnson R.C.
      • Mayadas T.N.
      • Frenette P.S.
      • Mebius R.E.
      • Subramaniam M.
      • Lacasce A.
      • Hynes R.O.
      • Wagner D.D.
      Blood cell dynamics in P-selectin-deficient mice.
      and other studies showing histamine-induced P-selectin facilitating neutrophil adhesion via CD11/CD18 integrin activation
      • Lorant D.E.
      • Patel K.D.
      • McIntyre T.M.
      • McEver R.P.
      • Prescott S.M.
      • Zimmerman G.A.
      Coexpression of GMP-140 and PAF by endothelium stimulated by histamine or thrombin: a juxtacrine system for adhesion and activation of neutrophils.
      and the development of allergic inflammation.
      • Dang B.
      • Wiehler S.
      • Patel K.D.
      Increased PSGL-1 expression on granulocytes from allergic-asthmatic subjects results in enhanced leukocyte recruitment under flow conditions.
      The significance of P-selectin in mediating leukocyte-endothelial cell interactions has been confirmed in patients with leukocyte adhesion deficiency (LAD II). These patients experience recurrent staphylococcal infections, and their neutrophils fail to roll and adhere adequately for lack of functional expression of sialyl Lewis X, a fucose-containing glycoconjugate ligand for P-, E-, and L-selectins.
      • Etzioni A.
      Defects in the leukocyte adhesion cascade.
      Identifying the mechanisms underpinning the regulation of P-selectin surface expression may therefore aid in development of new pharmaceutical approaches to combat allergic inflammation. A role for S1P in histamine-induced gene regulation of E-selectin and ICAM-1 was demonstrated by Shimamura et al,
      • Shimamura K.
      • Takashiro Y.
      • Akiyama N.
      • Hirabayashi T.
      • Murayama T.
      Expression of adhesion molecules by sphingosine 1-phosphate and histamine in endothelial cells.
      and it is our contention that the SK/S1P pathway in fact plays a critical role before gene regulation, with exocytosis of P-selectin occurring within minutes of exposure to histamine.
      Our results suggest that HUVECs exposed to histamine rapidly activate SK-1 and SK-2. To delineate the contribution of SK-1 versus SK-2 in this system, we used both broad-spectrum and specific SK inhibitors in in vitro and in vivo experiments. DMS is an inhibitor of both SK-1 and SK-2, but it also affects other lipid and protein kinases, including protein kinase C (PKC).
      • Pitman M.R.
      • Pitson S.M.
      Inhibitors of the sphingosine kinase pathway as potential therapeutics.
      In contrast, SKi is a more specific inhibitor. A recent report suggests that it specifically targets SK-1.
      • French K.J.
      • Schrecengost R.S.
      • Lee B.D.
      • Zhuang Y.
      • Smith S.N.
      • Eberly J.L.
      • Yun J.K.
      • Smith C.D.
      Discovery and evaluation of inhibitors of human sphingosine kinase.
      • Ren S.
      • Xin C.
      • Pfeilschifter J.
      • Huwiler A.
      A novel mode of action of the putative sphingosine kinase inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (SKI II): induction of lysosomal sphingosine kinase 1 degradation.
      Conversely, the inhibitor ABC294640 specifically targets SK-2.
      • French K.J.
      • Zhuang Y.
      • Maines L.W.
      • Gao P.
      • Wang W.
      • Beljanski V.
      • Upson J.J.
      • Green C.L.
      • Keller S.N.
      • Smith C.D.
      Pharmacology and antitumor activity of ABC294640, a selective inhibitor of sphingosine kinase-2.
      Our data from using these inhibitors suggest that only histamine-induced SK-1 activity is required for rapid surface expression of P-selectin on endothelial cells and neutrophil rolling events in vitro. Furthermore, extracellular S1P and the S1P1–3 receptors appeared to play no major role in the present study, which differs from the findings of Matsushita et al,
      • Matsushita K.
      • Morrell C.N.
      • Lowenstein C.J.
      Sphingosine 1-phosphate activates Weibel-Palade body exocytosis.
      who demonstrated that exposure of the human aortic endothelial cell line HAEC to 1 μmol/L S1P for 5 minutes caused release of von Willebrand factor, another protein stored preformed in Weibel-Palade bodies, and that 10 pmol/L of S1P injected intravenously into mice increased soluble P-selectin within 1 hour.
      The present study raises an alternative possibility, that intracellular second messengers modulated by S1P (eg, HDAC1/2, TRAF2, or prohibitin
      • Hait N.C.
      • Allegood J.
      • Maceyka M.
      • Strub G.M.
      • Harikumar K.B.
      • Singh S.K.
      • Luo C.
      • Marmorstein R.
      • Kordula T.
      • Milstien S.
      • Spiegel S.
      Regulation of histone acetylation in the nucleus by sphingosine-1-phosphate [Erratum appeared in Science 2009, 326:366].
      • Alvarez S.E.
      • Harikumar K.B.
      • Hait N.C.
      • Allegood J.
      • Strub G.M.
      • Kim E.Y.
      • Maceyka M.
      • Jiang H.
      • Luo C.
      • Kordula T.
      • Milstien S.
      • Spiegel S.
      Sphingosine-1-phosphate is a missing cofactor for the E3 ubiquitin ligase TRAF2.
      • Strub G.M.
      • Paillard M.
      • Liang J.
      • Gomez L.
      • Allegood J.C.
      • Hait N.C.
      • Maceyka M.
      • Price M.M.
      • Chen Q.
      • Simpson D.C.
      • Kordula T.
      • Milstien S.
      • Lesnefsky E.J.
      • Spiegel S.
      Sphingosine-1-phosphate produced by sphingosine kinase 2 in mitochondria interacts with prohibitin 2 to regulate complex IV assembly and respiration.
      ) may be involved. Clearly, the difference observed between the present findings and those of Matsushita et al
      • Matsushita K.
      • Morrell C.N.
      • Lowenstein C.J.
      Sphingosine 1-phosphate activates Weibel-Palade body exocytosis.
      requires further investigation in vitro and in vivo, using multiple approaches (including, but not limited to, the family of SK and S1P receptor knockout mice). In the present study, pretreatment of HUVECs with fingolimod caused a reduction in histamine-induced P-selectin expression and leukocyte rolling events. Fingolimod is an orally active immunomodulatory prodrug that recently gained U.S. Food and Drug Administration approval for treatment of multiple sclerosis,
      • Kappos L.
      • Radue E.W.
      • O'Connor P.
      • Polman C.
      • Hohlfeld R.
      • Calabresi P.
      • Selmaj K.
      • Agoropoulou C.
      • Leyk M.
      • Zhang-Auberson L.
      • Burtin P.
      FREEDOMS Study Group
      A placebo-controlled trial of oral fingolimod in relapsing multiple sclerosis.
      based on its ability to inhibit lymphocyte egress from lymph nodes and thymus.
      • Brinkmann V.
      • Billich A.
      • Baumruker T.
      • Heining P.
      • Schmouder R.
      • Francis G.
      • Aradhye S.
      • Burtin P.
      Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis.
      The mechanisms underpinning fingolimod inhibition of histamine-induced P-selectin expression and leukocyte rolling flux are still unknown, but likely are due to the ability of fingolimod to inhibit and degrade SK-1 in vitro.
      • Vessey D.A.
      • Kelley M.
      • Zhang J.
      • Li L.
      • Tao R.
      • Karliner J.S.
      Dimethylsphingosine and FTY720 inhibit the SK1 form but activate the SK2 form of sphingosine kinase from rat heart.
      • Lee W.J.
      • Yoo H.S.
      • Suh P.G.
      • Oh S.
      • Lim J.S.
      • Lee Y.M.
      Sphingosine mediates FTY720-induced apoptosis in LLC-PK1 cells.
      • Tonelli F.
      • Lim K.G.
      • Loveridge C.
      • Long J.
      • Pitson S.M.
      • Tigyi G.
      • Bittman R.
      • Pyne S.
      • Pyne N.J.
      FTY720 and (S)-FTY720 vinylphosphonate inhibit sphingosine kinase 1 and promote its proteasomal degradation in human pulmonary artery smooth muscle, breast cancer and androgen-independent prostate cancer cells.
      To provide additional definitive confirmation of the role of SK-1 in histamine-induced P-selectin expression in HUVECs, we attempted to use transient transfection with siRNA to knock down SK-1 expression; however, these experiments proved to be not technically feasible. A major limitation to working with P-selectin in primary HUVECs is that, after two or more passages, HUVECs lose their ability express preformed P-selectin.
      • Kameda H.
      • Morita I.
      • Handa M.
      • Kaburaki J.
      • Yoshida T.
      • Mimori T.
      • Murota S.
      • Ikeda Y.
      Re-expression of functional P-selectin molecules on the endothelial cell surface by repeated stimulation with thrombin.
      siRNA experiments necessarily involve additional passages, which precluded our ability to combine siRNA treatment with assessment of histamine-induced P-selectin mobilization in HUVECs. Nonetheless, our examination of in vivo responses in mice specifically lacking either SK-1 or SK-2 provided strong evidence supporting our hypothesis that SK-1 is critical to histamine-induced P-selectin up-regulation.
      Our in vivo studies showed that either pharmacological or genetic manipulation of SK-1 attenuates histamine-induced neutrophil rolling flux, which is critical for acute allergic inflammation. More specifically, we observed in WT mice that both SKi and fingolimod significantly attenuated histamine-induced neutrophil rolling flux. Consistent with SK-1 mediation of this process, Sphk1−/− mice exhibited significant resistance to histamine-induced neutrophil rolling flux, but Sphk2−/− mice did not. These findings differ from those of Michaud et al,
      • Michaud J.
      • Kohno M.
      • Proia R.L.
      • Hla T.
      Normal acute and chronic inflammatory responses in sphingosine kinase 1 knockout mice.
      who reported equivalent neutrophil numbers in the lavage fluid of both WT and Sphk1−/− mice in an inflammatory model of peritonitis using a 4-hour thioglycolate challenge.
      • Michaud J.
      • Kohno M.
      • Proia R.L.
      • Hla T.
      Normal acute and chronic inflammatory responses in sphingosine kinase 1 knockout mice.
      The divergence in these data may be attributable to the difference in the time courses of the responses investigated (ie, 5 to 10 minutes versus 4 hours) and the nature of the inflammatory stimuli (ie, histamine versus thioglycolate). We also showed that untreated WT, Sphk1−/−, and Sphk2−/− mice exhibited similar levels of baseline neutrophil rolling flux. Constitutive P-selectin expression in the lung, skin, intestine, mesentery, and cremaster muscle has been previously shown using the noninvasive dual radiolabeling antibody binding assay, so the finding is not the result of intravital microscopy intervention.
      • Hickey M.J.
      • Kanwar S.
      • McCafferty D.M.
      • Granger D.N.
      • Eppihimer M.J.
      • Kubes P.
      Varying roles of E-selectin and P-selectin in different microvascular beds in response to antigen.
      • Eppihimer M.J.
      • Wolitzky B.
      • Anderson D.C.
      • Labow M.A.
      • Granger D.N.
      Heterogeneity of expression of E- and P-selectins in vivo.
      Collectively, these data indicate that constitutive P-selectin expression in the cremaster muscle is SK independent, but that histamine-induced exocytosis of P-selectin expression is SK dependent.
      The physiological relevance of the differences in SK-1 and SK-2 activity levels with respect to allergy may be widespread,
      • Rivera J.
      • Proia R.L.
      • Olivera A.
      The alliance of sphingosine-1-phosphate and its receptors in immunity.
      and are yet to be fully elucidated. Experimentally, Pushparaj et al
      • Pushparaj P.N.
      • Manikandan J.
      • Tay H.K.
      • H'Ng S.C.
      • Kumar S.D.
      • Pfeilschifter J.
      • Huwiler A.
      • Melendez A.J.
      Sphingosine kinase 1 is pivotal for Fc epsilon RI-mediated mast cell signaling and functional responses in vitro and in vivo.
      showed both in vitro and in vivo that silencing SK-1 inhibited several mast-cell effector functions triggered by FcεRI engagement, whereas silencing SK-2 had no effect. However, there is still controversy concerning the different roles of SK-1 and SK-2 in mast-cell responses. Findings from a study using Sphk-deficient mice suggested that SK-2, and not SK-1, is more important for degranulation and cytokine or eicosanoid production by mast cells.
      • Olivera A.
      • Mizugishi K.
      • Tikhonova A.
      • Ciaccia L.
      • Odom S.
      • Proia R.L.
      • Rivera J.
      The sphingosine kinase-sphingosine-1-phosphate axis is a determinant of mast cell function and anaphylaxis.
      In addition, Zemann et al
      • Zemann B.
      • Urtz N.
      • Reuschel R.
      • Mechtcheriakova D.
      • Bornancin F.
      • Badegruber R.
      • Baumruker T.
      • Billich A.
      Normal neutrophil functions in sphingosine kinase type 1 and 2 knockout mice.
      showed that bone marrow-derived neutrophils from both Sphk1−/− and Sphk2−/− mice had normal functions of increasing intracellular Ca2+ and migration toward chemoattractants fMLP and C5a, compared with WT mice. Together, these studies suggest that the effects of SK isoforms may be cell-type specific.
      The prevalence of all types of allergies continues to rise across all age, sex, and racial groups. The Allergy and Asthma Foundation of America rating allergy as the third most common chronic disease among children.
      Chronic Conditions: A Challenge for the 21st Centrury.
      An understanding of the cellular and soluble mediators that are involved in allergic inflammation not only helps in elucidating the mechanisms of current treatments, but is also important for the identification of new therapeutic targets. Successful outcomes in future studies may establish SK as a therapeutic target to control histamine-induced allergic responses. More specifically, by targeting the early allergic response of neutrophil recruitment, we may be able to interfere in the initiation of chronic diseases triggered by allergens. Our understanding of this complex relationship might also reveal new opportunities for treatment of other diseases in which histamine is suggested to play a role (such as multiple sclerosis, rheumatoid arthritis, and psoriatic arthritis) but for which traditional antihistamines are generally regarded as ineffective.

      Acknowledgments

      We thank Michaelia Cockshell for preparing the endothelial cells and the staff and consenting donors at Women's and Children's Hospital and Burnside Memorial Hospital for collection of the umbilical cords.

      Supplementary data

      • Supplemental Figure S1

        Phosphorylated ERK-1/2 does not increase SK-1 protein levels. HUVECs were preincubated with ERK-1/2 pathway inhibitor (U0126; 20 μmol/L, 30 minutes) prior to histamine treatment (25 μmol/L, 5 minutes), cell lysis, and Western blotting for SK-1 protein. Quantified results are expressed as means ± SEM (n = 5).

      • Supplemental Figure S2

        Interactions between T cells and HUVECs after histamine activation. A: Untreated Jurkat cells (solid dark gray line) exhibit rapid shedding of L-selectin within 5 minutes of exposure to 25 μmol/L histamine (solid black line) as determined by flow cytometry. These levels are similar to that expressed by Jurkat cells treated with phorbol myristate acetate (PMA; 100 ng/mL, 30 minutes) (solid light gray line) and by untreated Jurkat T cells labeled with an isotype control antibody (dotted light gray line). Data are representative of three independent experiments. B: HUVECs were perfused without or with histamine (25 μmol/L, 2.5 minutes) prior to perfusion of Jurkat cells treated without or with histamine (25 μmol/L, 30 minutes) or freshly isolated neutrophils at 1 × 106 cells/mL. Data are expressed as rolling flux (means ± SEM) per FOV, with four to five FOV captured (n = 3). *P < 0.05 versus untreated (−).

      • Supplemental Video S1

        Parallel plate flow chamber video of HUVECs pretreated without or with a control antibody (10 μg, 30 minutes), P-selectin blocking antibody (10 μg, 30 minutes), or SK-1 inhibitor (SKi; 5 μmol/L, 10 minutes) before perfusion of histamine (25 μmol/L, 2.5 minutes) and freshly isolated human blood (5 minutes). Playback speed is 1×.

      • Supplemental Video S2

        Intravital videos from untreated controls, vehicle control, and mice treated with SKi or fingolimod prior to histamine superfusion (100 μmol/L, 5 minutes). Videos are 5-second images (corresponding to Figure 5A).

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