Isabella Fiorentino

Urokinase Induces Basophil Chemotaxis through a Urokinase Receptor Epitope That Is an Endogenous Ligand for Formyl Peptide Receptor-Like 1 and -Like 2

Basophils circulate in the blood and are able to migrate into tissues at sites of inflammation. Urokinase plasminogen activator (uPA) binds a specific high affinity surface receptor (uPAR). The uPA-uPAR system is crucial for cell adhesion and migration, and tissue repair. We have investigated the presence and function of the uPA-uPAR system in human basophils. The expression of uPAR was found at both mRNA and protein levels. The receptor was expressed on the cell surface of basophils, in the intact and cleaved forms. Basophils did not express uPA at either the protein or mRNA level. uPA (10−12–10−9 M) and its uPAR-binding N-terminal fragment (ATF) were potent chemoattractants for basophils, but did not induce histamine or cytokine release. Inactivation of uPA enzymatic activity by di-isopropyl fluorophosphate did not affect its chemotactic activity. A polyclonal Ab against uPAR inhibited uPA-dependent basophil chemotaxis. The uPAR-derived peptide 84–95 (uPAR84–95) induced basophil chemotaxis. Basophils expressed mRNA for the formyl peptide receptors formyl peptide receptor (FPR), FPR-like 1 (FPRL1), and FPRL2. The FPR antagonist cyclosporin H prevented chemotaxis induced by FMLP, but not that induced by uPA and uPAR84–95. Incubation of basophils with low and high concentrations of FMLP, which desensitize FPR and FPRL1, respectively, but not FPRL2, slightly reduced the chemotactic response to uPA and uPAR84–95. In contrast, desensitization with WKYMVm, which also binds FPRL2, markedly inhibited the response to both molecules. Thus, uPA is a potent chemoattractant for basophils that seems to act through exposure of the chemotactic uPAR epitope uPAR84–95, which is an endogenous ligand for FPRL2 and FPRL1.

Expression and functions of the vascular endothelial growth factors and their receptors in human basophils.

Angiogenesis is a multistep complex phenomenon critical for several inflammatory and neoplastic disorders. Basophils, normally confined to peripheral blood, can infiltrate the sites of chronic inflammation. In an attempt to obtain insights into the mechanism(s) underlying human basophil chemotaxis and its role in inflammation, we have characterized the expression and function of vascular endothelial growth factors (VEGFs) and their receptors in these cells. Basophils express mRNA for three isoforms of VEGF-A (121, 165, and 189) and two isoforms of VEGF-B (167 and 186). Peripheral blood and basophils in nasal polyps contain VEGF-A localized in secretory granules. The concentration of VEGF-A in basophils was 144.4 ± 10.8 pg/106 cells. Immunologic activation of basophils induced the release of VEGF-A. VEGF-A (10–500 ng/ml) induced basophil chemotaxis. Supernatants of activated basophils induced an angiogenic response in the chick embryo chorioallantoic membrane that was inhibited by an anti-VEGF-A Ab. The tyrosine kinase VEGFR-2 (VEGFR-2/KDR) mRNA was expressed in basophils. These cells also expressed mRNA for the soluble form of VEGFR-1 and neuropilin (NRP)1 and NRP2. Flow cytometric analysis indicated that basophils express epitopes recognized by mAbs against the extracellular domains of VEGFR-2, NRP1, and NRP2. Our data suggest that basophils could play a role in angiogenesis and inflammation through the expression of several forms of VEGF and their receptors.

Basophils Infiltrate Human Gastric Mucosa at Sites of Helicobacter pylori Infection, and Exhibit Chemotaxis in Response to H. pylori-derived Peptide Hp(2–20)

Basophils, which are normally confined to the circulation, can migrate to sites of allergic inflammation. Using the specific mAb, BB1, we detected basophil infiltration of the gastric mucosa of Helicobacter pylori-infected patients affected by moderate and severe gastritis. Basophils were not found in H. pylori-free individuals or in subjects with mild gastritis. The H. pylori-derived peptide, Hp(2–20), was a potent basophil chemoattractant in vitro, whereas the control peptide, Hp1, was ineffective. Basophils from peripheral blood of healthy volunteers expressed mRNA for the formyl peptide receptors, N-formyl-peptide receptor (FPR), FPR-like (FPRL)1, and FPRL2. Preincubation of basophils with FMLP or Hp(2–20) caused complete desensitization to a subsequent challenge with homologous stimulus. Incubation of basophils with a low concentration of FMLP, which binds with high affinity to FPR, but not to FPRL1 or FPRL2, did not affect the chemotactic response to Hp(2–20). In contrast, a high concentration of FMLP, which binds to FPRL1 and FPRL2, reduced the chemotactic response to Hp(2–20). The FPR antagonist, cyclosporin H, prevented chemotaxis induced by FMLP, but not by Hp(2–20). Hp(2–20) could be responsible, at least in part, for basophil infiltration of the gastric mucosa of H. pylori-infected patients presumably through the interaction with FPRL1 and FPRL2.

Neopterin induces pro‐atherothrombotic phenotype in human coronary endothelial cells

Summary.  Background: Inflammation plays a pivotal role in atherothrombosis. Recent data indicate that serum levels of neopterin, a marker of inflammation and immune modulator secreted by monocytes/macrophages, are elevated in patients with acute coronary syndromes and seem to be a prognostic marker for major cardiovascular events. The aim of the present study was to determine whether neopterin might affect the thrombotic and atherosclerotic characteristics of human coronary artery endothelial cells (HCAECs). Methods and results: In HCAECs, neopterin induced TF‐mRNA transcription as demonstrated by real time polymerase chain reaction and expression of functionally active tissue factor (TF) as demonstrated by procoagulant activity assay, and of cellular adhesion molecules (CAMs) as demonstrated by FACS analysis, in a dose‐dependent fashion. These neopterin effects were prevented by lovastatin, a HMG‐CoA reductase inhibitor. Neopterin‐induced TF and CAMs expression was mediated by oxygen free radicals through the activation of the transcription factor, nuclear factor‐kappa B (NF‐κB), as demonstrated by electrophoretic mobility shift assay and by suppression of CAMs and TF expression by superoxide dismutase and by NF‐κB inhibitor, pyrrolidine‐dithio‐carbamate ammonium. Conclusions: These data indicate that neopterin exerts direct effects on HCAECs by promoting CAMs and TF expression and support the hypothesis that neopterin, besides representing a marker of inflammation, might be an effector molecule able to induce a pro‐atherothrombotic phenotype in cells of the coronary circulation.

HIV-1 Envelope gp41 Peptides Promote Migration of Human FcεRI+ Cells and Inhibit IL-13 Synthesis Through Interaction with Formyl Peptide Receptors

We evaluated the effects of synthetic peptides (2017, 2019, 2020, 2021, 2023, 2027, 2029, 2030, 2031, and 2035) encompassing the structure of HIV-1MN envelope gp41 on both chemotaxis of human basophils and the release of preformed mediators (histamine) and of cytokines (IL-13). Peptides 2019 and 2021 were potent basophil chemoattractants, whereas the other peptides examined were ineffective. Preincubation of basophils with FMLP or gp41 2019 resulted in complete desensitization to a subsequent challenge with homologous stimulus. Incubation of basophils with low concentration (5 × 10−7 M) of FMLP, which binds with high affinity to N-formyl peptide receptor (FPR), but not to FPR-like 1, did not affect the chemotactic response to a heterologous stimulus (gp41 2019). In contrast, a high concentration (10−4 M) of FMLP, which binds also to FPR-like 1, significantly reduced the chemotactic response to gp41 2019. The FPR antagonist cyclosporin H inhibited chemotaxis induced by FMLP, but not by gp41 2019. None of these peptides singly induced the release of histamine or cytokines (IL-4 and IL-13) from basophils. However, low concentrations of peptides 2019 and 2021 (10−8–10−6 M) inhibited histamine release from basophils challenged with FMLP but not the secretion caused by anti-IgE and gp120. Preincubation of basophils with peptides 2019 and 2021 inhibited the expression of both IL-13 mRNA, and the FMLP-induced release of IL-13 from basophils. These data highlight the complexity of the interactions between viral and bacterial peptides with FPR subtypes on human basophils.

Human urotensin II induces tissue factor and cellular adhesion molecules expression in human coronary endothelial cells: an emerging role for urotensin II in cardiovascular disease

Summary.  Background: Human urotensin II is an 11‐aminoacid peptide with a controversial role in the human cardiovascular system. Indeed, urotensin effects on vascular reactivity and in heart failure are well documented, while its potential role in the pathophysiology of athero‐thrombosis is still unknown. This study investigates the effects of urotensin on tissue factor (TF) and VCAM‐1/ICAM‐1 expression in human coronary endothelial cells (HCAECs). Methods and results: Urotensin induced TF‐mRNA transcription as demonstrated by real time PCR and expression of TF that was functionally active as demonstrated by procoagulant activity assay. In addition, urotensin induced expression of VCAM‐1 and ICAM‐1 as demonstrated by FACS analysis. VCAM‐1 and ICAM‐1 were functionally active because they increased leukocyte adhesivity to HCAECs. Urotensin‐induced expression of TF and of VCAM‐1/ICAM‐1 was mediated through the Rho A‐activation of the transcription factor, NF‐κB, as demonstrated by EMSA. Indeed, lovastatin, an HMG‐CoA reductase inhibitor, by modulating the Rho activation, and NF‐κB inhibitors, suppressed the urotensin effects on TF and CAMs. Conclusions: Data of the present study, although in vitro, describe the close relationship existing between urotensin II and athero‐thrombosis, providing for the first time support for the view that this peptide might have not only vasoactive functions but it might be an effector molecule able to induce a pro‐atherothrombotic phenotype in cells of the coronary circulation. Although future studies are required to clarify whether these mechanisms are also important in the clinical setting, this report supports an emerging new role for urotensin II in the pathogenesis and progression of cardiovascular disease.

HMG-CoA reductase inhibitors reduce nicotine-induced expression of cellular adhesion molecules in cultured human coronary endothelial cells.

Background: Smoking predisposes to the development of atherosclerosis and of its complications. The mechanisms responsible for these effects are not completely understood. We have investigated whether nicotine might promote a proatherosclerotic state in human coronary endothelial cells (HCAECs), studying the role of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors in preventing these phenomena. Methods and Results: Real-time PCR showed that nicotine induced a dose-dependent increase in mRNA levels for vascular cellular adhesion molecule-1 (VCAM-1)/intercellular adhesion molecule-1 (ICAM-1). Fluorescent-activated cell sorting analysis showed that nicotine induced expression of functionally active VCAM-1/ICAM-1, since they increased leukocyte adherence to HCAECs. Oxygen free radicals, Rho A and nuclear factor ĸB (NF-ĸB) play a pivotal role in modulating these effects. Indeed, nicotine caused oxygen free radical production as well as activation of Rho A and NF-ĸB pathways, evaluated by malondialdehyde levels, pulldown assay and by electrophoretic mobility shift assay, respectively. Superoxide dimutase, Rho A (Y-27639) and NF-ĸB inhibitors (pyrrolidine dithiocarbamate ammonium, Bay 11-7082) suppressed nicotine effects on CAM expression. HMG-CoA reductase inhibitors prevented these nicotine-mediated effects by inhibiting free radical generation and by modulating activation of Rho A and NF-ĸB pathways. Conclusions: Nicotine promotes CAM expression on HCAECs, shifting them toward a proatherosclerotic state. These effects might explain, at least in part, the deleterious cardiovascular consequences of cigarette smoking. HMG-CoA reductase inhibitors play an important role in preventing these phenomena.

Thanks to the Reviewers

CHRISTOPHER ADAMSON MICHAEL P. ALLEN WILLIAM BAILEY STEVE BALKIN RICHARD E. BALL JOYCE BARAKETT ROBERT A. BEAUREGARD E. M. BECK HOWARD S. BECKER LAWRENCE BENNETT BENNETT BERGER LEONARD BERKEY MICHAEL BETZ EGON BITTNER LEONARD BLUMBERG PHILIP BLUMSTEIN ROBERT BOGDEN EDNA BONACICH PEG BORTNER VERN BULLOUGH LEONARD CAIN KITTY CALAVITA JOHN CARSLEY JOAN CASSELL PAUL CHALFANT RANDALL COLLINS MARK COLVIN JOHN A. CONLEY ELAINE CUMMING ARLENE KAPLAN DANIELS JON DARLING JOAN DEBARDELEBEN CHARLES DERBER STEVEN DEUTSCH IRWIN DEUTSCHER PAUL DIMAGGIO JOAN EAKIN WILLIAM EATON SUSAN ECKSTEIN SHELDON EKLAND-OLSON ROBERT EMERSON PAULA S. ENGLAND DAVID M. ERMANN DARYL EVANS WILLIAM FALK SAMUEL FARBER ROB FAULKNER JOE FEAGIN ROSLYN FELDBERG SANDY FELDHEIM MARK FISHMAN NANCY FRANK ELIOT FREIDSON PETER FREITAG EDGAR Z. FRIEDENBERG WILLIAM FRIEDLAND SAMUEL FRIEDMAN JOHN GALLIHER DAVID GARTMAN GILBERT GEIS JAMES GESCHWENDER DON C. GIBBONS PEGGY GIORDANO DANIEL GLASER FRED GOLDNER ROBERT A. GORDON WALTER GOVE SUSAN GRAY DAVID GREENBERG ALLEN D. GRIMSHAW EDWARD GROSS BRUCE HACKETT JOHN HAGAN RICHARD F. HAMILTON SUE KIEFER HAMMERSMITH SHARON HARLAN CLAYTON HARTJEN JAMES HENSLIN JOHN P. HEWITT BARBARA HEYL SALLY T. HILLSMAN LYNDA LYTLE HOLMSTROM RANDY HODSON ALLEN HORWITZ JOAN HUBER DREW HUMPHRIES ALLEN W. IMERSHEIN JAMES INCIARDI DAVID JACOBS GARY JENSEN CAROLE JOFFE JOHN JOHNSON PAUL JOSEPH RACHEL KAHN-HUT DEBRA KALMUSS JACK KATZ RONALD KESSLER JOSEPH A. KOTARBA RONALD KRAMER NANCY KUTNER BARBARA R. LASLETT PAT LAUDERDALE RONALD LAWSON JEFFREY LEITER RHONDA F. LEVINE CYRIL LEVITT ELLIOT LIEBOW CLARENCE LO ULI LOCHER MARGARET LOCK HELENA Z. LOPATA DAVID E. LOPEZ DAVID LUCKENBILL PETER LYMAN LARRY LYON STEVE LONGSTAFF CHARLES McCAGHY JOHN D. McCARTHY SCOTT G. McNALL LINDA C. MAJKA GERALD MARKLE JOHN MARKOFF CORA MARRETT TONY MASI DOUGLAS MAYNARD ROBERT MEIER JANET E. MICKISH TERANCE MIETHE S.M. MILLER BETH MINTZ MERRY ANN MORASH DAVID L. MORGAN CAROL KIAPERMAN MORROW CHARLES C. MOSCOS JR MARTHA MYERS JOANE NAGEL DOROTHY NELKIN MARGARET K. NELSON LINDA B. NILSON STEPHEN NORLAND MELVIN OLIVER MARVIN OLSEN SUSAN OLZAK ANN L. PAGE TOBY PARCEL DOROTHY PAWLUCH HAROLD E. PEPINSKY CHARLES PERROW ROBERT PERRUCCI SUZANNE PETERS KAREN J. PETERSON MICHAEL PETRUNIK MARK PEYROT GERALD PLATT ALPHONSO PINKNEY HENRY PONTELL ROBERT C. PRUS RICHARD QUINNEY NICOLE FISCHER RAFTER PRUDENCE RAINS DONNA RANDALL STEVEN RANDALL JOSEPH RANKIN RICHARD RATCLIFF PAMELA RICHARDS JAMES RICHARDSON RAY RIST JACK ROACH JANET ROACH JIM ROBBINS E. BURKE ROCHFORD JOSEPH W. ROGERS ROBIN ROOM RACHEL ROSENFELD BARBARA KATZ ROTHMAN LILLIAN RUBIN JOSEPHINE A. RUGGIERO WILLIAM A. RUSHING SHERYL RUZEK SASKIA SASSEN-KOOB SANDRA P. SCHOENBERG WILLIAM SHAFFIR NANCY S. SHAW CLIFFORD D. SHEARING JAMES F. SHORT JR. ROBERTA SIMMONS DOUGLAS SMITH MICHAEL SMITH DAVID A. SNOW LEE SODERSTROM JACK W. SPENCER STEVEN SPITZER DARRELL J. STEFENMEIER RONALD TAYLOR JIM THOMAS ROBERT THOMAS GARY TIEDEMAN KATHLEEN TIERNEY RONALD TROYER JAMES D. UNNEVER BERT USEEM PAULINE VAILLANCOURT ARTHUR J. VIDICH DIANA VAUGHAN EDWARD J. WALSH VIVIENNE WALTERS CATHERINE WATSON J. ALLEN WHITT SIDNEY WILLHELM JUDITH G. WITTNER PETER YIN MAYER N. ZALD DAVID ZARET